Advances in Enzymology and Related Areas of Molecular Biology, Volume 67

515 Pages·1993·10.918 MB·English

ADVANCES IN ENZYMOLOGY
AND RELATED AREAS OF
MOLECULAR BIOLOGY
Volume 67
LIST OF CONTRIBUTORS
JEFFT . BOLIN, Biological Sciences, Purdue University, West Lafayette. IN 47907.
GEORGEW . GOKEL,D epartment of Chemistry, University of Miami, Coral Gables,
FL 33 124.
JEFFREY1 . GORDOND, epartment of Molecular Biology and Pharmacology, Wash-
ington University School of Medicine, St. Louis, MO 631 10.
MARKR . HARPEL.B. iology Division, Oak Ridge National Laboratory, Oak Ridge,
TN 378314077,
FREDC. HARTMANB,i ology Division, Oak Ridge National Laboratory, Oak Ridge.
TN 37831-8077.
SEYMOUKRA UFMANLa, boratory of Neurochemistry, National Institute of Mental
Health, Bethesda. MD 20892.
RADHAG . KKISHNAD,e partment of Biochemistry and Molecular Biology, University
of Texas Medical School, Houston, TX 7722s.
CHARLEAS. MCWHERrEK, Monsanto Company. St. Louis, MO 63198.
T. VANCEM ORGANC.e nter for Metalloenzyme Studies, University of Georgia, Ath-
ens, GA 30602.
LEONARED. MORYXNSON,C enter for Metalloenzyme Studies, University of Georgia,
Athens, GA 30602.
DAVIDA . RUDNICKD,e partment of Molecular Biology and Pharmacology. Wash-
ington University School of Medicine. St. Louis. MO 631 10.
LANCEC . SEEFELD?C, enter for Metalloenzyme Studies, University of Georgia. Ath-
ens. GA 30602.
FINNW OLD, Department of Biochemistry and Molecular Biology, University of
Texas Medical School, Houston, TX 77225.
RAYW u, Section of Biochemistry, Molecular and Cell Biology, Cornell University,
Ithaca, NY 148.53.
ADVANCES IN ENZYMOLOGY
AND RELATED AREAS OF MOLECULAR BIOLOGY
Founded by F. F. NORD
Edited by ALTON MEISTER
CORNELL UNIVERSITY MEDICAL COLLEGE
NEW YORK, NEW YORK
VOLUME 67
WILEY
1993
AN INTERSCIENCES PUBLlCATION
JOHN WILEY & SONS, INC. .
New York Chichester Brisbane Toronto Singapore
9
This text is printed on acid-free paper.
Copyright 0 1993 by John Wiley & Sons. Inc.
All rights reserved. Published simultaneously in Canada.
Reproduction or translation of any part of this work beyond
that permitted by Section 107 or 108 of the 1976 United
States Copyright Act without the permission of the copyright
owner is unlawful. Requests for permission or further
information should be addressed to the Permissions Department.
John Wiley & Sons, Inc.. 605 Third Avenue. New York, NY
10158-001 2.
This publication is designed to provide accurate and
authoritative information in regard to the sub,ject
matter covered. It is sold with the understanding that
the publisher is not engaged in rendering legal. accounting,
or other professional services. If legal advice or other
expert ascistance is required, the services of a competent
professional person should be sought.
Library of Congress Catalog Card Number: 41-9213
ISBN 0-471-58279-4
Printed in the United States of America
10 9 8 7 6 5 4 3 2
CONTENTS
Chemical and Genetic Probes of the Active Site of D-
Ribulose-1 $Bifphosphate CarboxylaselOxygenase: A
Retrospective Based on the Three-Dimensional
Structure .................................................. 1
Fred C. Hartman and Mark R. Harpel
Phenylalanine Hydroxylating System ...................... 77
Seymour Kaufman
Post-translational Modification of Proteins ................ 265
Radha G. Krishna and Finn Wold
The Role of Metal Clusters and MgATP in Nitrogenase
Catalysis ................................................... 299
Leonard E. Mortenson, Lance C. Srefeldt,
T. Vance Morgan and Jeff T. Bolin
Myristoyl CoA: Protein N-Myristoyl-transferase ......... 375
David A. Rudnick, Charles A. McWherter,
George W. Gokel and Jeflrey I. Gordon
Development of Enzyme-Based Methods for DNA
Sequence Analysis and Their Applications in the
Genome Projects .......................................... 43 1
Ray Wu
Author Index ................................................ 469
Subject Index ................................................ 50 1
V
ADVANCES IN ENZYMOLOGY
AND RELATED AREAS OF
MOLECULAR BIOLOGY
Volume 67
Advances in Enzymology and Related Areas of .!MolecularB iology, Volume 67
Edited by Alton Meister
Copyright 0 1993 by John Wiley & Sons, Inc.
CHEMICAL AND GENETIC PROBES OF THE
ACTIVE SITE OF D-RIBULOSE-~,~-
BISPHOSPHATE CARBOXYLASEl
OXYGENASE: A RETROSPECTIVE BASED
ON THE THREE-DIMENSIONAL
STRUCTURE
By FRED C. HARTMAN and MARK R.
HARPEL, Biology Division, Oak Ridge National
Laboratory, Oak Ridge, TN
CONTENTS
I. Introduction
11. Reaction Pathway
A. Carboxylation
B. Oxygenation
C. Kinetic Mechanism, Rate-Limitations, and Alternate Substrates
D. Side Reactions
E. Minimal Requirements for Rubisco Catalysis
Ill. Crystal Structures
A. Features Common to Lz and L& Enzymes
9. L8S8 Enzymes
C. The Active Site
IV . Active Site Characterization with General Reagents
V. Active Site Characterization with Affinity Labels
VI. Chemical Properties of the Two Active-Site Lysines Identified with Affinity
Labels and Their Inter-Residue Distance
VII. General Considerations of Site-Directed Mutagenesis and Characterization
of Rubisco Mutants
VIII. Chemical Rescue
IX . Site-Directed Mutagenesis Guided by Chemical Modification and
Comparative Sequences
A. Validation (or Invalidation) of Presumed Importance of Residues
B. Function of Active-Site Residues
C. Active-Site Location and Intersubunit Interactions
X. Structure-Guided Mutagenesis
A. Substitution of a Phosphate-Binding Site
B. The Invisible Base
1
2 FRED C. HARTMAN AND MARK R. HARPEL
C. Intersubunit, Electrostatic Interactions
XI. Carboxylase/Oxygenase Specificity
XII. Concluding Remarks
Acknowledgments
References
I. Introduction
Prior to knowledge of its function, D-ribulose-1 ,5-bisphosphate
carboxylase/oxygenase (Rubisco) (E.C. 4.1.1.39) had been purified
from tobacco and denoted as fraction protein (1). Not until ten
Z
years later was this high-abundance protein identified as Rubisco
after detection of its carboxylation activity during photosynthesis
and its purification based on the monitoring of that activity. Pi-
oneering studies from the laboratories of M. Calvin, B. L. Horecker,
and E. Racker conclusively proved that Rubisco catalyzes carbox-
ylation of D-ribulose- 1,5-bisphosphate (RuBP) to form two molar
equivalents of 3-phospho-~-glycerate( PGA), thereby fulfilling the
crucial requirement for photosynthetic fixation of carbon dioxide
with the net synthesis of carbohydrate (for an historical account,
see ref. 2). Another twenty years elapsed before Rubisco was shown
to possess oxygenase in addition to carboxylase activity, whereby
RuBP undergoes oxidative degradation to phosphoglycolate and
PGA (Fig. I). This startling and profound codiscovery by the groups
of Ogren (3) and Tolbert (4, 5) placed Rubisco at the interface of
two competing metabolic pathways in which the single enzyme ini-
tiates both photosynthetic carbon reduction and photorespiration.
Oxygenase activity of Rubisco appears to be a physiologically
functionless but unavoidable consequence of the chemical proper-
ties of the 2,3-enediol(ate) of RuBP, the initial intermediate in both
reaction pathways. The relative reactivity of the enediol(ate) toward
COz and O2 dictates the partitioning ratio between the carboxylation
and oxygenation pathways. Three-quarters of the phosphoglycolate
formed by oxidation of RuBP is returned to the Calvin cycle by the
energy-requiring glycolate pathway (6). Despite this salvage path-
way, net C02-fixation by C3 plants (plants in which the Rubisco-
containing mesophyll cells are in direct contact with the external
CHEMICAL AND GENETIC PROBES OF RUBISCO 3
0
/I
c -0-
CO, + HO, 2 HC-OH /o- 2 H+
I
H,C-OP\
0 I1 0
I1 /0- 0
H,C-OP,
I 0-
HC?-=OOH Enz
HC-OH Mg"
I /o-
h,C-OP, 0
0II 0 c0II- o- c11 -o-
H,CI- OP\ /o i HCI -OH /o- + 2 H+
02 6 0 H,C-OP, -
II 0
0
Figure 1. Stoichiornetries of overall reactions catalyzed by Rubisco.
atmosphere) is diminished about one-third by photorespiration. Ap-
preciation of the negative impact of Rubisco's oxygenase activity
on plant growth and hence the potential advantages of elevating the
carboxylase/oxygenase ratio by genetic or chemical manipulation
has certainly stimulated interest in Rubisco. However, many addi-
tional factors have contributed to the enormous attention focused
on Rubisco by biochemists, enzymologists, X-ray crystallographers,
molecular biologists, geneticists, plant physiologists, and agrono-
mists. These factors will be briefly considered.
Rubisco may be viewed as a cornerstone of life; it links the in-
organic and organic pools of carbon on our planet and provides the
only globally significant route for the net synthesis of carbohydrate
from atmospheric COz. The enzyme also provides the major chem-
ical route for removing COz from the atmosphere, a fact of potential
pertinence to amelioration of the greenhouse effect. Because the
carboxylation reaction is rate-limiting in photosynthetic carbon as-
similation, Rubisco is a prime determinant of biomass yield. The
slow rate (k,,, = 2-5 s-') probably explains the preponderance of
Rubisco, which approaches 50% of the total soluble protein in green
leaves or an active-site concentration of 3 mM in chloroplasts! Un-
doubtedly, Rubisco is the world's most abundant protein. Besides
the low k,,, , other mechanistically intriguing features include the
absence of cofactors typically associated with oxygenases (Fez ,
+
4 FRED C. HARTMAN AND MARK R. HARPEL
Cu2+,f lavins) and an unusual obligatory activation that entails re-
action of C02, distinct from substrate COz, with a lysyl €-amino
group to form a carbamate, which is stabilized by the essential Mg2+
ion (7, 8):
0 0
II I1
2c 0 2 Mg2 +
Enz-NH2 Enz-NH-C-0- Enz-NH-C-O---Mg*'
G
c02
Under physiological conditions of [H'l, [Mg2+], [COZI, and
[RuBP], the equilibrium favors the nonactivated form of the enzyme
(9). This perplexity was resolved by the discovery of another chlo-
roplastic protein, denoted activase, which facilitates carbamylation
in vivo (for a review, see ref. 10). The mechanism is incompletely
understood but involves the release of inhibitory phosphorylated
ligands (including RuBP) from the noncarbamylated protein. The
ATP-requirement of activase explains, in part, light-mediated acti-
vation of Rubisco; however, proteins of the thylakoid membrane are
also involved (11).
In some plant species, Rubisco activity is regulated by a tight-
binding inhibitor, 2-carboxyarabinitol- 1 -phosphate, that accumu-
lates in the dark (reviewed in ref. 12). A specific phosphatase relieves
this inhibition, possibly following activase-assisted release of the
bound inhibitor from the active site. This phosphatase is sensitive
to the levels of sugar phosphates in the cell and is inactive in the
absence of reduced thiols, suggesting that the light-dependence of
this inhibition may be thioredoxin-mediated (see ref. 13 and citations
therein).
Because of several characteristics, Rubisco is an excellent model
for exploring fundamental problems in plant molecular biology. The
high abundance of the enzyme's cognate mRNA facilitates inves-
tigations of regulation of gene expression and its response to photon
flux (14-16). Most species of Rubisco contain eight large (L) and
eight small (S) subunits; the L subunits are chloroplastic encoded,
whereas the S subunits are nuclear encoded. Both types of subunits
are synthesized as larger precursors that are processed by specific
proteases prior to assembly of the active L8Ss species (17-21). Thus,