STEROLS AND BILE ACIDS


New Comprehensive Biochemistry
Volume 12

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Urrecht

ELSEVIER
AMSTERDAM NEW YORK OXFORD
*

*


Sterols and Bile Acids

Editors

HENRY DANIELSSON

a

and JAN SJOVALL

Department of Pharmaceutical Biochemistiy, University of Uppsala, Uppsala
(Sweden) and Department of Physiological Chemistiy, Karolinska Institutet,
Stockholm (Sweden)

a

1985
ELSEVIER
Amsterdam - New York Oxford
*


1985. Elsecier Science Publishers B.V (Biomedical Division)
All nghts reserved. No part of ths publication may be reproduced. stored in a retrieval system or
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of the U.S.A., should be referred to the publisher.
ISBN for the series: 0-444-80303-3
ISBN for the volume: 0-444-80670-9
Published by :
Fl\evier Science Publishers B.V. (Biomedical Division)
P.0 Box 211
loo0 AE Amsterdam

(The Netherlands)
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Library of Congress Cataloging-in-Publication Data
Main entry under title.
Sterols and bile acids

(New comprehensive biochemistry: v. 12)
Includes bibliographies and index.
1 . Sterols--1Metabolism.2. Bile acids-Metabolism.
I. Danielsson. Henry. 11. Sjovall. Jan. 111. Series.
[DNLM: 1. Bile Acids and Salts--metabolism. 2. Sterols
--metabolism. W1 NE372F v.12 / QU 95 S8391
QD415.N48 VOI.12 574.19’2 s 1574.19’2431185-20620
/QB752.S75]
ISBN 0-444-80670-9 (U.S.)

Printed in The Netherlands


Preface
Sterols are essential components of all eukaryotic cells. Their function is structural, and by being precursors of hormones and bile acids they exert a regulatory
function on metabolic processes. Cholesterol and its metabolism are of importance
in human disease. Although the mechanisms are largely unknown, it can be surmised
that abnormalities in the metabolism of sterols and bile acids are associated with
cardiovascular disease and gallstone formation. Steroid hormones are vital for man,

animals and plants. Disturbances in their production can have deleterious consequences.
T h s volume of New Comprehensive Biochemistry is entitled Sterols and Bile
Acids. It includes fourteen chapters written by prominent scientists in the field. The
large volume of material in the field of sterols and bile acids has necessitated a
limitation of the areas covered. Chapters on steroid hormones have been excluded
since this field requires a volume of its own. In spite of this it has not been possible
to produce a book of some few hundred pages. Efforts have been made to condense
the contributions of the individual authors, but the wealth of important information
is such that a further reduction in size would seriously affect the value of the
chapters to the reader.
It may be argued that there are important gaps in the contents of this volume. For
instance, full discussions of the role of compartmentation of sterols and their
metabolism, of the dynamics of cholesterol balance, etc. are lacking. We as editors
take full responsibility for ths. Our only excuse is that the material contained in the
volume is already at the limit of what can be accommodated in a volume of New
Comprehensive Biochemistry.
Although we have tried to make terminology, abbreviations, etc. reasonably
uniform it will be apparent that there are differences between chapters, but hopefully not within a chapter. We have felt it more important to let the individualities of
the authors be expressed.
It is our conviction that the eminent contributions in this volume, for which we
are very grateful to our colleagues, will be of the value they deserve for all scientists
in the field of sterol and bile acid research.
Uppsala and Stockholm
September 1985

Henry Danielsson
Jan Sjovall


This Page Intentionally Left Blank



H. Danielsson and J. Sjovall (Eds.), Sterols and Bile Acids
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

Contents
........

v

Chapter I
Biosynthesis of cholesterol
Hans
C. filling and Liliane T. Chayet (Salt Lake City and Santiago) . . . . .
-

1

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................

1

.....
.....
.....

3
4


................

8

.................................
................

11

I.
11.
111.

Acetyl-CoA acetyl transferase (EC 2.3.1.9)
1. Cellular location . . . . . . . . . . . . . . . .
2. Enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

IV.

2. Enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V.
2. Mechanism

VI.
VII. Phosphomevalonate kinase (EC 2.7.4.2)
VIII.
IX.

1. Enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . .

...........................
........
.....

12
13

.....

14

.................................................
...........................
.....

21

X.
3. Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Termination

XI.

Squalene synthetase . . . .

20

......................

............................

24

XII.
1. Enzymology . . . . .
2. Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.....

28


XIII. The conversion of lanosterol to cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Sterol function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. General aspects of enzymology . . . . . . . . . . . . . . . . . . . . . .
.......................................
3. The pathway . . . . . . . .
4. Decarbonylation at (2-14
.......................................
5. Decarboxylation at C-4 . . . . .
........................
6. Introduction of As . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 2
Control mechanisms in steroi uptake and biosynthesis
John F. Gill Jr., Peter J. Kennelly and Victor W. Rodwell (West tafayette) .
1.


11.

Control of sterol uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 . Plasma lipoproteins , . . . . , , , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. Normal plasma lipoproteins, 41 - b. Abnormal plasma lipoproteins,
Chemically modified lipoproteins, 44 2. Cellular mechanisms of cholesterol uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. The LDL receptor and receptor-mediated endocytosis, 45 - b. Other receptor-mediated lipoprotein uptake mechanisms, 48 - c. Receptor
ndent cholesterol uptake, 51
....................
3. Regulation of receptor-mediated sterol uptake . . . . .
____..._......._
4. Diseases related to receptor-mediated cholesterol upta
a. Homozygous FH, receptor-negative and receptor-defective, 54 - b. Heterozygous
FH. 56 - c. Homozygous FH. internalization-defective, 56 - d. Wolman syndrome, 56
.- e. Cholesterol ester storage disease, 56 Control of sterol biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Mammalian HMG-CoA reductase
..........................
a. Rate-limiting step in sterol bi
b. Distribution, 57 - c. Diurnal
rhythm and developmental pattern, 58 - d. Regulation of HMG-CoA reductase protein
level. 59 - e. Modulation of HMG-CoA reductase activity, 62 2. Other sites of control . .
..........................
a. Early sites of control. 65 - b. Later sites of control, 66 ~

31
31
31
32
32

34
36
37
37

41
41
41

44

52

_54
__.

57
57

65

67
61

Chapier 3
Participution of sterol carrier proteins in cholesterol biosynthesis, utilization and
intracellulur transfer
Terence J. Scallen and George V. Vahouny (Albuquerque and Washington)
I.
11.


Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sterol carrier protein, (SCP,)
1 ..................................
1. Purification and characteri
.......................
2. Substratespecificity . . .
3. Participation of SCP, in
.................................
squalene to sterol . . . . .
4. Properties of SCP, . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73
13
74
74
74
75
75


ix
111.

IV.

Sterol carrier protein, (SCP,) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Participation of SCP, in cholesterolbiosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. General remarks, 77 - b. Purification and characterization of SCP,, 77 - c.
Substrate specificity,78 - d. Kinetic studies, 78 - e. Anti-SCP, IgG, 792. Participation of SCP, in cholesterolutilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a. Cholesterol esterification, 80 - b. Cholesterol 7a-hydroxylase,81 3. Participation of SCP, in intracellular cholesterol transfer . . . . . . . . . . . . . . . . . . . . . . .
a. SCP, is required for cholesterol transport from adrenal lipid droplets to mitochondria,
82 - b. Identification of adrenal SCP,, 84 - c. SCP, facilitates the translocation of
cholesterol from the outer to the inner mitochondrial membrane, 85 4. Comparisoqof SCP, with other low molecular weight proteins . . . . . . . . . . . . . . . . . . .
a. Fatty-acid-binding protein (FABP), 87 - b. Nonspecific phospholipid exchange
protein, 90 The participation of sterol carrier proteins in intracellular cholesterol metabolism . . . . . . . .

77
77

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91
91

Chapter 4
Biosynthesis, function and metabolism of sterol esters
Alan Jones and John Glomset (Seattle) . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
11. Distribution and physical properties of cholesterolesters .
111. Enzymes and proteins that mediate the formation, transp
esters . . . . . . . . . . .
1. Acyl-CoA :cholesterol acyltransferase(EC 2.3.1.26) . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Cholesterol ester hydrolase (EC 3.1.1.13) . .
a. Acid CEH, 101 - b. Neutral CEH, 102 3. Lecithin :cholesterolacyltransferase(EC 2.3.1.43)
4. Plasma cholesterolester transfer protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Physiology of choles
.............

1. Plasma lipoprotein cholesterol esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Tissue cholesterolesters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. Fibroblasts, 111 - b. Cells that form steroid hormones, 112 - c. Macrophages, 112
- d. Hepatocytes, 113 Cholesterol sulfate . . . . . . . . . . . .
V.
VI. Cholesterol esters and disease . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . .
VII. Conclusion . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

80

82

87

91

95
95
95
98
98
101
103
106
107
107
110

113

114
116
116

Chapter 5
Cholesterol absorption and metabolism by the intestinal epithelium
Eduard F. Stange and John M. Dietschy (Dallas) . . . . . . . . . . . . . . . . . . . 121
I.
11.
111.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Absorption of cholesterol . . . .
.............
Intestinal cholesterolsynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...
1. Methodology.. . . . . . . . . . .
2. Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

121
123
125
125
126


X

1V.


3 . Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Intestinal lipoprotein uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..
...
1. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Localization . . . . . . . . . . .
................

3. Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Function . . . . . . . . . . . . . . . . . . . . . . . . . . .
Intestinal cholesterol esterification . . . . . . . . . . . . . . . . . . . . . . . . .
V.
1 . Acyl-CoA: cholesterol acyltransferase (ACAT) . . . . . . . . . . . . . . . . . .
a . Methodology, 136 - b . Localization. 137 - c . Regulation, 138 - d .
2. Cholesterol esterase of pancreatic origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a. Methodology . 140 - b . Localization 140 - c . Regulation, 140 - d . Function. 140
VI . Origin of the cholesterol
tinal lymph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...........................................
1. Total cholesterol mass
1..............................
2. Newly synthesized ch
V11 . Compartmentalization of cholesterol in the enterocyte . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Cliupter 6
Cholesterol and hiomemhrane structures
D . Chapman. Mary T.C. Kramers and C.J. Restall (London) . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Occurrence of sterols . . . . . . . .
.....................
Cholesterol-phospholipid interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The effects of cholesterol upon mem
The effects of cholesterol on cellular functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cholesterol exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.............
.............
VII . The experimental manipulation of cholesterol . . . . . . . . . . . .
VIII . Cholesterol and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Liver disease . . . . . . . . . . . . . . .
..
2 . Familial hypercholesterolaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Ageing and atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Muscular dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Cholesterol and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
rx . Cholesterol evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
X . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

11.
111.
IV .
V.
VI .


Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 7
Bios.ynthesis of plunr sterols
T.W . Goodwin (Liverpool) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
11.

I11 .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Formation of cycloartenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alkylation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

128
132
132
132
133
134
135
136
136
140
141
141
142
144

146
146

151
151
151
152
159
161
162
163
164
164
164
165
166
166
166
168
169
170
170

175
175
175
178


xi

Steps other than those involving methylation . . . . . . . . . . . . . . . . . . . . . . . . . . .
...............................
1. Demethylation . . . . . . . . . . . . . . . . . .
...
2. Isomerization of A* + A5 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Insertion of the A2’ double bond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V.
Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Steroid hormones . . . . . . . .
........................................
VII. Cardiac glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VIII. Sapogenins .
IX. Ecdysteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Steroid alkaloids . . . . . . . .
X.
IV.

2. As hormones

184
184
187
188
189
190
190
192
192
193
194

194
196
196
196
196
197

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

198

.

.............._..

XIII. Sterol esters . .
XIV. Sterol glycosides

Chapter 8
Structures, biosynthesis and function of sterols in invertebrates
Nobuo Ikekawa (Tokyo) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
I.

Sterols in marine invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction . . .
2. Sterols in Porifera
3. Sterols in Coelenterata . . . . . . . .

6. Sterols in Mollusca and others. . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Side chain modification of sterols in marine invertebrates. . . . . . . . .

1. Dealkylation of phytosterol
2. Inhibitors of phytosterol met
3. Structure requirement of ster

............_.....
................................
owth and development . . . . . . . . . . . . . . . . . . . . .

2. Ecdysteroids isolated from insects and their biosynthesis . . . .
3. Ecdysone metabolism and biological activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199
199
201
204
207
209
210
212
213
213
217
219
219
219
220
222
225

225

Chapter 9
Mechanism of bile acid biosynthesis in mammalian liver
Ingemar Bjorkhem (Stockholm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
~

I.
11.

111.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Formulation of the sequence of reactions in the biosynthesis of bile acids . . . . . . . . . . . . . .
Cholesterol 7a-hydroxylase . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Assay of cholesterol 7a-hydroxylase in liver microsomes . . . . . . . . . . . . . . . . . . . . . . .
2. Substrate specificity and physiological substrate for cholesterol 7a-hydroxylase . . . . . . .

23 1
233
237
237
239


XU

3. Mechanism of 7a-hydroxylation of cholesterol and experiments with purified enzyme
components
..............................................

240
I V. Conversion of 7a-hydroxycholestero1 into 7a-hydroxy-4-cholesten-3-one
. . . . . . . . . 244
V.
12a-Hydroxylation
..............................................
245
VI . Saturation of the A4
bond . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
246
VII . Reduction of the 3-0x0 group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
247
VIII . 26-Hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
247
1. Microsomal 26-hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
248
2. Mitochondria1 26-hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
249
into 3a,7a.l2c~-trihydroxy-S~-cholestanoic
IX . Conversion of 5~-cholestane-3a.7a.l2a,26-tetrol
acid . .
..............................................
251
X.
Conversion
hydroxy-5~-cholestanoicacid into cholic acid
..
252
XI . Conjugation of the carboxylic group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
253

XI1 . Formation of allo bile acids in the liver . . . . . . . . . . . . . .
. . 255
1 . Conversion of 5p-bile acids to allo bile acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
255
2. Conversion of choiestanol into allo bile acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
255
3 . Conversion of 7a-hydroxy-4-cholesten-3-one
into allo bile acids . . . . . . . . . . . . . . . . . . 257
XI11. Species differences and alternative pathways in the biosynthesis of bile acids . . . . . . . . . . . 257
XIV . Inborn errors of metabolism in bile acid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . .
261
1 . Cerebrotendinous xanthomatosis . . . . . . . . . . . . . . . .
. . 261
2 . Zellweger's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
263
. . 264
XV . Regulation of the overall biosynthesis of bile acids . . . . . . . . . . . .
1. Feedback regulation of bile acid biosynthesis .
.........................
264
2 . Relation between cholesterol 7a-hydroxylase and HMG-CoA reductase . . . . . . . . . . . . . 266
3. Possible mechanisms for regulation of cholesterol 7a-hydroxylase activity . . . . . . . . . . . 268
XVI . Regulation of the ratio between cholic acid and chenodeoxycholic acid . . . . . . . . . . . . . . .
270
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 10
Bile ulcohols and primitive bile acids
Takahiko Hoshita (Hiroshima) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.
I1 .

Introduction . . . . . . . . . . . . .
........................
Occurrence and structure of bile alcohols in lower vertebrates . . . . . . . . . . . . . . . . . . . . . .
I11. Occurrence and structure of primitive bile acids in lower vertebrates . . . . . . . . . . . . . . . . .
I V. Occurrence and structure of bile alcohols in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . .
Occurrence and structure of primitive bile acids in mammals . . . . . . . . . . . . . . . . . . . . . .
V.
V I . Metabolism of bile alcohols and primitive bile acids in mammals . . . . . . . . . . . . . . . . . . .
V l i . Metabolism of bile alcohols and primitive bile acids in lower vertebrates . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter I 1
Metabolism of bile acids in liver and extrahepatic tissues
William H . Elliott (St. Louis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Enterohepatic circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Subcellularlocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Transport and hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

272
272

279
279
279

285
289
291
293
296
299

303
303
303
304
306


11.

Conjugation

. . . . . . . . . . . . . . .> . . . . . . . . . . . . . . . . . . . . .
....._.............
.. .. .....

a. Preparation and analyses of CoA derivatives, 307 - b. Bile acid : CoA synthetase (bile
acid CoA ligase) and bile acid CoA : glycine/taurine-N-acyltransferase,307 - c.
Metabolism, 308 2. Sulfates . . . . . . . . . . . . . .
...................
3. Glucuronides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV.
V.

VI.

1. 3a-Hydroxylation . . . . . . . . . . . . . . . . . . . . . .
....... ..
2. 6P- and 6a-hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. 7a- and 7P-hydroxylation . . . . . . . . . . . . . . . . . . . . . ,
4. 12a-Hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . .
.. .. . . .
5. 16a-Hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydroxylation in the side chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Oxidoreduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extrahepatic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Brain . . . . . . . . . . . . . . . . . . .
.................
2. Cecum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

............................
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.....................

Chapter 12
Metabolism of bile acids in intestinal microflora
Phillip B. Hylemon (Richmond) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
11.
111.
IV.
V.


VI.

306
306

309
310
310
311
311
313
315
315
316
317
320
320
320
321
324
324
325

331

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Composition of intestinal microflora . . . . . . . . . .
. . .... ..
Deconjugation.. . . . . . . . . . . . . . . . . . . . . . . . .
... .. . . .

Desulfation
.......................................
Epimerizati
..........................
1. 30- and 38-hydroxysteroid dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. 6a- and 6P-hydroxysteroid dehydrogenase
..........
3. 7a-Hydroxysteroid dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. 7P-Hydroxysteroid dehydrogenase . . . . . . . . .
5. 12a-Hydroxysteroid dehydrogenase . . . . . . . .
7a- and 7P-dehydroxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

331
332
333
334
334
335
335
336
336
337
338

Acknowledgement
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

341
342


Chapter 13
Physical-chemical properties of bile acids and their salts
Martin C. Carey (Boston)
-I.
11.

111.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Molecular structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Chemistry.. . . . . .
2. Hydrophilic-hydrop
.......................................
3. Miscellaneous physi
Crystalline structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
....................
1. Bile acids and their derivatives . . . . . . . . . .

345
345
345
348
349
352
352


XIV

2. Bile salt hydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. “Choleic acids” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..
I V . Surface physical chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Bileacids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Bile salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Mixed monolayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V.
Bulk aqueous physical chemistrv . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. pH-solubility relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Bile acids -.-solubility behavior . . . . . . . . . . . . . . . . . . . . . . .
.........
3. Temperature-solubility relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V I . Micelle formation in aqueous solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Critical micellar concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a . Methods of determining bile salt CMC values. 372 - b . Influence of variation in
physical-chemical conditions. 372 - c. Inventory of published data. 373 2. Micellar size and polydispersity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a . Unconjugated bile salts 374 - b . Glycine-conjugated bile salts. 374 - c. Taurineconjugated bile salts 375 - d . Polydispersity. 375 3. Micellar shape and hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Micellar structures . . .
...........................
5 . Micellar charge and co
n binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Rates of exchange of micellar and intermicellar components . . . . . . . . . . . . . . . . . . . . .
7 . Critical and non-critical self-association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII . Reverse micelle formation in non-aqueous media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Bile acid esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Bile acids and salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Bile salts within the hydrophobic domai
omes and membranes . . . . . . . . . . . .
V I I I . Mixed micelle formation . . . . . . . . . . . . .
............................

1. Bile salt- hydrocarbon micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Bile salt-insoluble amphiphile micelles . . . . . . . . .
.....
......
3. Bile salt-swelling amptuphile micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Bile salt-soluble amphiphile micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Bile salt-swelling amphiphile-insoluble amptuphile micelles . . . . . . . . . . . . . . . . . . . .
6 . Mixed micelle-unilamellar vesicle transition . . . . . .
................
7 . Native bile and intestinal content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

352
358
359
359
360
362
364
365
369
370
372
372

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.........

397
397


.

.

313

375
377
378
379
379
382
382
383
384
388
388
389
390
393
394
395
396

Chupter 14
Roles of bile acids in intestinal lipid digestion and absorption
B. Borgstrom. J.A. Barrowman and M . Lindstrom (Lund) . . . . . . . . . . . . . 405
I.


11.

111.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
......
1. Historical remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Brief outline of intestinal lipid digestion and absorption . . . . . . . . . . . . . . . . . . . . . . . .
Roles o f bile salts in the intestinal lumen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. What is the physical state of the lipids in the intestinal contents? . . . . . . . . . . . . . . . . . .
2. Bile salts are bad emulsifiers! . . . . . . . . . . . . . . . . . . . .
.........
3. What is the importance of bile salts for the function of the lipolyt
4 . What is the composition of the luminal contents after a fat meal?
Roles of bile salts in transport of lipolytic products from lumen of cytosol through mucosal
diffusion barriers and plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Methods employed in the study of lipid absorption . . . . . . . . . . . . . . . . . . . . . . . . . . .

405
405
405
406
406
407
407
409
410
410



xv

IV .
V.

VI .

2. Which are the barriers for lipid transport from lumen to cytosol?
..............
3 . What factors determine the rate of transport across the intestinal
sion barriers? . . . .
4. What is the role of micellar solubilization for intestinal lipid absorption? . . . . . . . . . . . .
5 . Absorption of lipids from non-micellar phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Roles of bile salts in intracellular events in lipid absorption . . . . . . . . . . . . . . . . . . . . . . .
Roles of bile salts in the absorption of specific lipid classes . . . . . . . . . . . . . . . . . . . . . . . .
1. Triglycerides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Phospholipids . . .
..............
3 . Cholesterol . . . . .
4. Fat-soluble vitamins
..............
5 . Other nonpolar compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Are bile salts necessary for lipid absorption? . . . . . . .

411
413
414
416
417
419

419
419

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

422

SubjectIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

420
421

427


This Page Intentionally Left Blank


H. Danielsson and J. Sjovall (Eds.), Sterols and Bile Acrdr
1985 Elsevier Science Publishers B.V. (Biomedical Division)

0

CHAPTER 1

Biosynthesis of cholesterol
HANS C. RILLING a and LILIANA T. CHAYET
a

Department of Biochemistry, University of Utah School of Medicine,

Salt Lake City, UT 84108 (U.S.A.), and
Departamento de Bioquimica, Facultad de Ciencias Basicas y
Farmaceuticas, Universidad de Chile, Santiago (Chile)

(I) Introduction
It is not possible to write a comprehensive review of cholesterol biosynthesis in
the space allotted nor is it our desire to do so. Consequently, we have been selective
as to what has been included and the list of references is far from exhaustive. We
hope that no one will be offended by our choices. In addition, where appropriate,
the stress has been on enzymes from liver. There are several recent and comprehensive reviews on cholesterol biosynthesis. Two books, one by Nes and McKean [l]
and another by Gibbons, Mitropoulos and Myant [2] provide excellent and current
reviews on the biosynthesis and function of cholesterol. In addition, about half of
the chapters in a book edited by Porter and Spurgeon [3] deal with selected aspects
of the biochemistry of sterologenesis and, taken as an aggregate, provide a comprehensive review of the subject. Also Schroepfer has published recently two reviews on
cholesterol biosynthesis in Annual Reviews of Biochemistry [4,5].
Cholesterol is primarily restricted to eukaryotic cells where it plays a number of
roles. Undoubtedly, the most primitive function is as a structural component of
membranes. Its metabolism to bile acids and the steroid hormones is relatively
recent in the evolutionary sense. In this chapter, the pathway of cholesterol biosynthesis will be divided into segments which correspond to the chemical and
biochemical divisions of this biosynthetic route. The initial part of the pathway is the
3-step conversion of acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA).
The next is the reduction of this molecule to mevalonate, considered to be the
rate-controlling step in the biosynthesis of polyisoprenoids. From thence, a series of
phosphorylation reactions both activate and decarboxylate mevalonate to isopentenyl pyrophosphate, the true isoprenoid precursor. After a rearrangement to the
allylic pyrophosphate, dimethylallyl pyrophosphate, a sequence of 1'-4 con1


2


densations (head-to-tail) * between the allylic and homoallylic pyrophosphates leads
to the synthesis of prenyl pyrophosphates whch are requisite for the synthesis of the
higher polyprenols such as cholesterol, dolichol, and coenzyme Q. For the synthesis
of cholesterol, the polymerization is halted at the sesquiterpene level. Two molecules
of the farnesyl pyrophosphate thus formed condense in a
manner (head-tohead) producing presqualene pyrophosphate. After a reductive rearrangement of the
carbon skeleton to squalene, an epoxidation leads to the formation of 2,3-oxidosqualene whch then cyclizes to lanosterol. The final stages of sterologenesis involve
the removal of 3 methyl groups from lanosterol and the migration and reduction of
double bonds to give cholesterol. In higher organisms, this pathway is primarily
restricted to the liver, small intestine, kidney, and endocrine organs. While other
classes of cells do maintain the enzymes for this set of reactions, they depend upon
the liver as a source of sterol.
Very early in the investigation of sterol biosynthesis it was established that acetate
was the primary precursor. In 1942 Bloch and Rittenberg found that deutero-acetate
could be converted to cholesterol in the intact animal in high yields [7]. This was in
accord with the earlier observation of Sonderhoff and Thomas that the nonsaponifiable lipids from yeast (primarily sterol) were heavily labeled by the same substrate
181. Degradation of the sterol molecule in the laboratories of both Bloch and Popjak
showed that all of the carbon atoms of cholesterol were derived from acetate and
that the labeling pattern of methyl and carboxyl carbons originating from acetate
indicated that the molecule was isoprenoid in nature 191. It was apparent then that
sterols have as their fundamental building block, acetate, a molecule that resides at
the center of intermediary metabolism.

(II) Source of acetyl-CoA
The synthesis of fatty acids and sterols in the liver cytosol depends upon a
common pool of acetyl-CoA. This was demonstrated by Decker and Barth in a series
of experiments utilizing perfused rat liver [lo]. Lipid synthesis was measured by
incorporation of tritium from [ 'H]H,O. They used ( - )-hydroxycitrate to inhibit
ATP-dependent citrate lyase and measured radioisotope incorporation into fatty
acids and sterols as a function of the concentration of this inhibitor. A parallel

decrease in incorporation into these two products was found as the concentration of
( - )-hydroxycitrate in the perfusate was increased. Contrastingly, if radioisotopic
acetate was used as the substrate in the perfusing medium, this inhibitor had
relatively little effect on the rate of sterologenesis, a result that would be expected if
the natural source of acetate was from the action of the cytoplasmic citrate lyase.
Their experiments also demonstrated that the ratio of fatty acid synthesis to sterol
synthesis in the liver of fed rats is about 10 : 1.
* The nomenclature used is described in ref. 6, p. 163.


3

The beginning stages of fatty acid as well as cholesterol biosynthesis are cytoplasmic processes. The initial substrate for both pathways is acetyl-CoA which is
generated in the mitochondria primarily from glycolysis via pyruvate and pyruvate
dehydrogenase or by the p-oxidation of fatty acids. Since mitochondrial membranes
are impermeable to coenzyme A derivatives, other derivatives of acetate must be
utilized to move the acetyl unit from the mitochondria to the cytoplasm. The major
pathway involves citrate as the carrier. Citrate generated in the mitochondrion from
acetyl-CoA and oxaloacetate moves to the cytosol, a process that is facilitated by the
dicarboxylate transport system. In the cytosol, the citrate cleavage enzyme utilizes
ATP and CoA to convert citrate to acetyl-CoA and oxaloacetate. The acetyl group is
used for lipogenesis whle the oxaloacetate is reduced to malate by NADH. Malate is
then oxidized to pyruvate and CO, by NADP and the malic enzyme. This step
affords NADPH-reducing equivalents for both lipogenesis and sterologenesis. Either
malate or pyruvate can re-enter the mitochondrion.
Acetoacetate is another vehicle for transporting acetyl groups into the cytoplasm.
This molecule, one of the end products of ketone body synthesis, is free to diffuse
from the mitochondrion. When in the cytoplasm it can be activated to acetoacetylCoA by an ATP-dependent acetoacetyl-CoA synthetase. Edmonds’ group has shown
that the activity of this enzyme parallels the rate of cholesterologenesis in the livers
of animals given a variety of dietary regimes [ll]. Their data also indicate that this

pathway furnishes as much as 10% of the carbon required for cholesterol biosynthesis.
Several other pathways have been postulated for the transport of acetyl units
from the mitochondrion to the cytoplasm. One entails carnitine as a transporter, as
it is for fatty acids, the other invoked free acetate. It is unlikely that either of these is
significant [12].

(III)Acetyl-CoA ucetyl transferuse (EC 2.3.1.9)
The first enzyme in the pathway is acetoacetyl-CoA thiolase which catalyzes the
condensation of two molecules of acetyl-CoA.

0

II

2 CH,- C -S-CoA@CH,-

0

II

0

II

C -CH,- C -S-CoA

+ CoASH

(I) Cellular location
There are two discrete locations of enzymes for the biosynthesis of acetoacetyl-CoA

and HMG-CoA; one is mitochondrial and serves the purpose of generating ketone
bodies (acetoacetate and 3-hydroxybutyrate).The other is cytoplasmic and provides
precursors for isoprene units for the biosynthesis of cholesterol and other terpenoids.


4

Early in the study of the enzymology of the biosynthesis of HMG-CoA, there was
some confusion as to whether these processes were physically separated withm the
cell. Studies by Lane and his collaborators [13] and by others [14] clearly indicated a
duality of locus. The cytosolic enzyme, purified from avian liver [13], was found to
have a molecular weight of 1.7 x lo5 with 4 apparently identical subunits (41000 by
SDS gel electrophoresis). The cytosolic enzyme constituted 70% of total thiolase
found in chicken liver.
( 2 ) Enzymology

The equilibrium position of this reaction is 6 X lop6 which is quite unfavorable
for the synthesis of acetoacetyl-CoA. However, as will be pointed out below, when it
functions in conjunction with the next enzyme in the sequence, synthesis is favored.
The turnover number in the forward direction (as written) was found to be 1770
while that of the reverse was 54000. The enzyme from mitochondria has two
electrophoretically distinct forms, and the cytoplasmic enzyme could be clearly
distinguished from the two mitochondria1 proteins by the difference in their isoelectric points. Middleton surveyed many tissues from rat as well as selected tissues from
ox and pigeon for both the cytoplasmic and the mitochondria1 enzymes. The highest
levels of the cytoplasmic enzyme were found in the liver, adrenal, brain of the
neonate. and ileum [14,15]. There was an obvious positive correlation between sterol
biosynthetic capacity and the distribution of this enzyme. The mitochondrial enzyme
was found predominantly in heart, ludney, and liver. The cytosolic enzyme has also
been highly purified from rat liver [16].
(3) Mechanism


Kinetic analysis indicated that the mechanism is ping-pong with an acyl enzyme
intermediate [ 171. Since Lynen had shown earlier that -SH-directed reagents inhibit
the enzyme [18], it was assumed that an acyl-S-enzyme was an intermediate in the
reaction. Suicide substrates for this enzyme have been prepared and tested against
the protein isolated from heart mitochondria. The substrate analogs 4bromocrotonyl-. 3-pentenoyl-, 3-butynoyl-, and 2-bromoacetyl-CoA all progressively
and irreversibly inhibited the enzyme. Thus, 3-acetylenic CoA esters are effective
site-specific inhibitors of t h s enzyme and a mechanism to account for this is shown
in Fig. 1 . This inactivation resulted in the formation of a stable tho1 ether with the
enzyme. Other enzymes with putative sulfhydryl groups in the catalytic site behave
in a similar manner with acetylenic substrate analogs [19].
The enzyme could be inactivated by NaBH, in the presence of either acetoacetylCoA or acetyl-CoA. This observation strongly suggests that the reaction is through a
Claisen condensation with an amine as the base and with an enzyme substrate
ketimine as an intermediate [20]. A mechanism was postulated for the reaction as
indicated in Fig. 2.
The stereochemistry of acetoacetyl-CoA thiolase was examined by utilizing


5

Fig. I. The mechanism by which acetylenic analogs irreversibly inhibit acetyl-CoA acetyl transferase

s

y

N

COASH


CH3COSCOA

l-2
-

m
SH
NH2

Fig. 2. A mechanism for the reaction catalyzed by acetyl-CoA acetyl transferase.

(2S,3S)-3-hydro~y[2-*H,,~H~]butyryl-CoA
as substrate. It was cleaved by the enzyme and the resulting radioactive acetate examined for its chirality by standard
procedures. The results demonstrated that the cleavage reaction (and presumably the
condensation reaction) proceeds with inversion of the methylene group that becomes
the methyl group on cleavage [21].

(IV) 3-Hydroxy-3-methylglutaryl-CoAsynthetase (EC 4.1.3.5)
(I) Cellular location
3-Hydroxy-3-methylglutaryl-CoAsynthetase, like the enzyme that precedes it,


6

enjoys two subcellular locals.

0

II


CH3- C -S-CoA

0

II

+ CH3- C -CH,-

0

II

C -S-CoA
0

+HO-

II

OH

I

C -CH,-C-CH,-

0

I1

C -S-CoA


+ CoASH

I
CH,
In the mitochondrion it participates in ketone body synthesis, while as a cytoplasmic
enzyme it functions in cholesterol biogenesis [22].

( 2 ) Enzymology
This enzyme has been extensively studied by Lane and his collaborators utilizing
preparations from chicken liver as well as rat liver [22]. With chicken liver preparations, a number of forms of the enzyme were detected and purified. One was
associated with the mitochondrial fraction and was presumably the enzyme of
ketogenesis. Other. multiple forms were recovered from the cytosol. One of these was
immunoiogically cross-reactive with the mitochondrial enzyme. Since this form of
the cytosolic enzyme was also detected in tissues that do not have mitochondrial
HMG-CoA synthetases. such as brain and heart. it seemed unlikely that they were
dealing with a proteolytic artifact or a product precursor relationship between the
different proteins. The other three cytosolic synthetases were immunologically
related. The most abundant, synthetase 11, constituted more than 60% of the
cytosolic activity. This protein has a molecular weight of about 100000 and is
comprised of two subunits of identical molecular weight. Dissociation into monomers was easily accomplished by increasing the ionic strength. The other two
cytoplasmic forms could be derived from the predominant form presumably by a
minor modification such as a proteolytic cleavage or removal of a phosphate residue.
The level of the cytoplasmic proteins but not the mitochondrial form of the
synthetase was governed by the dietary regime of the birds. Cholesterol feeding,
which diminishes cholesterol synthesis, suppressed the activity level of the enzyme.
Inclusion of cholestyramine in the diet, which elevates cholesterol synthesis, enhanced the activity of the enzyme. Unlike avian liver, rat liver cytosol contains a
single HMG-CoA synthetase, suggesting that the multiple enzymes recovered from
chicken liver are artifacts. The rat liver cytosolic enzyme is subject to repression by
cholesterol feeding as well as by fasting; both procedures cause an 80% reduction in

activity within 24 h. Also, since cholestyramine caused an enhanced level of the
enzyme, the authors concluded that this enzyme might play a regulatory role in
cholesterol biosynthesis [22].


7

Because of the poor equilibrium position of acetoacetyl-CoA synthetase, they
tested the effect of the combined presence of acetoacetyl-CoA synthetase and
HMG-CoA synthetase on the equilibrium between acetyl-CoA and HMG-CoA. At
equilibrium slightly more than half of the acetyl-CoA had been converted to
product. The position of the equilibrium is governed by the expression
- [ HMG-CoA] [ CoAI2

Kapp-

[ acetyl-CoAI3

The KaPP determined .was 1.33. Thus, the overall equilibrium for the combined
reactions favors cholesterol biosynthesis. An improved preparation of HMG-CoA
synthetase from chicken liver mitochondria has been recently reported [23].

(3) Mechanism
The mechanism of this reaction was determined utilizing homogeneous protein
isolated from mitochondria [24]. The enzyme catalyzes an exchange of acetyl groups
between acetyl-CoA and several sulfhydryl-containing acceptors. The most effective
acceptor was dephospho-CoA, with an exchange rate 50% of normal condensation.
Cysteamine, the CoA analog used by Lynen to study fatty acid biosynthesis, also
participated in the exchange reaction but at 25% of the normal reaction. The enzyme
also catalyzes a slow hydrolysis of acetyl-CoA. These observations, plus the fact that

the exchange reactions followed zero-order kinetics, led to the postulate that an
enzyme-S-acetyl intermediate was involved in the reaction. Earlier, Stewart and
Rudney had shown that the thiol ester carbonyl of acetyl-CoA gave rise to the free
carboxyl of HMG-CoA [25], and it was postulated that the functionality on the
enzyme responsible for hydrolysis was the group involved in acyl transfer.
Brief incubation of the enzyme at 0°C with radioactive acetyl-CoA led to the
formation of acyl enzyme which could be isolated by chromatography on Sephadex
[24]. The enzyme-substrate complex was then reacted with acetoacetyl-CoA with the
concomitant formation of HMG-CoA. Further studies indicated that the functional
group on the enzyme that accepted the acetyl residue was a cysteine sulfhydryl.
4’-Phosphopantetheine is known to accept acyl residues, but it was not found in this
protein. The stoichiometry for acetylation was 0.7 acetyl groups per mole of enzyme;
since it is a dimeric protein with apparently identical subunits, this observation is
surprising. Thus, it is possible that the subunits perform different functions; for
example, one could be regulatory. It is interesting to note that both the thiolase and
HMG-CoA synthetase utilize acyl enzyme intermediates in their catalytic mechanisms.
In a later publication, another enzyme-bound intermediate involved in the
reaction mechanism of HMG-CoA synthetase was reported [26]. On the assumption
that acetylation of the enzyme by acetyl-CoA was the initial step in the reaction
sequence, they felt that condensation with the second substrate, acetoacetyl-CoA,


8
1 ) CH3-CO-SCOA + HS-Enz

CH,-CO-S-Enz

+

COASH


,CH,-CO-SCOA

2)C H 3 - C O - C ~ 2 - C O - S C ~+ ~CH3-CO-S-Enz e H y C

HO,

,CH~-CO-SCOA

c

3)
CH/

+ HzO
‘CH2-CO-S-Enz

-

/ \
CH3 CH2-CO-S-Enz

HO,

,CH2-CO-SCOA
C
CH: ‘CHZ-CO~H

Enz-SH


i

Fig. 3. A mechanism for the reaction catalyzed by 3-hydroxy-3-methylglutaryl-CoA
synthetase.

would lead to the formation of HMG-CoA-S-enzyme as a second enzyme-bound
intermediate. When acetylated enzyme was mixed with acetoacetyl-CoA at low
temperatures in the presence of a mixture of ethanol and glycerol, this intermediate
could be demonstrated. Perchloric acid was required to cleave the enzyme substrate
adduct, indicating that the linkage was covalent. Pronase digestion of the intermediate gave N-HMG-cysteine. Since S-to-N migration occurs under these conditions, they concluded that the bonding to the enzyme was through the sulfur of
cysteine. An isotope dilution method was used to evaluate the relative kinetic
constants for the formation and utilization of the acetyl-S-enzyme moiety. With this
technique the necessary requirements were met for the existence of covalent enzyme-substrate intermediate. The sequence of reactions leading to the formation of
HMG-CoA are shown in Fig. 3.
The enzyme demonstrates partial selectivity for its acyl substrate since both
acetyl-CoA and propionyl-CoA will react to give an acyl-S-enzyme species. However, only the acetyl-enzyme will react with acetoacetyl-CoA [23].The enzyme will
also bind other acyl groups such as a rather bulky spin-labeled CoA derivative,
3-carboxy-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl-CoA,
very tightly but not covalently in the acetyl-accepting site [27]. Interestingly, the enzyme will accept the
thioether analog of acetoacetyl-CoA, 3-oxobutyl-CoA, to give as product a thioether
analog of HMG-CoA. Because of the covalent linkage of one substrate and of
product to the enzyme, a ping-pong mechanism would be anticipated and has been
demonstrated for the mitochondria1 enzyme (281.

(V)3-Hydrox~-3-methyiglutaryl-CoA
reductase (EC I.I.I.34)
The enzymes so far described are identical, with respect to the reactions catalyzed,
to the enzymes that make ketone bodies. Since these pathways are physically
separated within the cell, it would seem that regulation of sterol synthesis could
reside in this region of the pathway. However, the key regulatory step in cholesterol

biosynthesis is HMG-CoA reductase (Chapter 2). A book on this protein has been
published recently [29],and among others Qureshi and Porter have also reviewed
this enzyme [12].Comments on this protein will be restricted to noting a few salient
features that will enable the reader to progress without referring to other sources.