University of Oulu

Protein crystallographic studies to understand the reaction mechanism of enzymes : α-methylacyl-CoA racemase and argininosuccinate lyase

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Author: Bhaumik, Prasenjit1,2
Organizations: 1University of Oulu, Faculty of Science, Department of Biochemistry
2University of Oulu, Biocenter Oulu
Format: ebook
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 2.6 MB)
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Language: English
Published: 2006
Publish Date: 2006-05-26
Thesis type: Doctoral Dissertation
Defence Note: Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kuusamonsali (Auditorium YB210), Linnanmaa, on June 6th, 2006, at 12 noon
Reviewer: Doctor Anette Henriksen
Professor Reijo Lahti


Enzymes catalyze chemical changes in biological systems. Therefore, to understand the chemistry of living systems, it is important to understand the enzyme structure and the chemistry of the enzyme’s functional groups which are involved in catalysis. In this study, structure and function relationships of two enzymes, (1) α-methylacyl-CoA racemase from Mycobacterium tuberculosis (MCR) and (2) argininosuccinate lyase from Escherichia coli (eASL) have been studied using X-ray crystallography. The main focus of this study has been understanding the structure-function relationship of MCR. The eASL has been crystallized from a highly concentrated sample of purified recombinant α-methylacyl-CoA racemase in which it occurred as a minor impurity.

The structure of eASL has been solved using molecular replacement at 2.44 Å resolution. The enzyme is a tetramer, but in this crystal form there is a dimer in the asymmetric unit. Each active site is constructed from loops of three different subunits. One of these catalytic loops, near residue Ser277 and Ser278, has been disordered in the previous structures of active lyases, but is very well ordered in this structure in one of the subunits due to the presence of two phosphate ions in the respective active site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex and therefore this structure has provided new information on the reaction mechanism of this class of enzymes.

α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of α-methyl-branched CoA esters. An Amacr homologue from the eubacteria Mycobacterium tuberculosis, referred to as MCR, was taken as a model protein. MCR was purified, crystallized and the structure of unliganded protein was determined at 1.8 Å resolution using the MIRAS procedure. The structure shows that the enzyme is an interlocked dimer.

To understand the reaction mechanism and the mode of substrate binding, several crystallographic binding studies were done using both wild type MCR and mutant H126A MCR crystals. In particular, the structures of the wild type MCR-complexes with (R, S)-ibuprofenoyl-CoA (1.85 Å), (R)-2-methylmyristoyl-CoA (1.6 Å) and (S)-2-methylmyristoyl-CoA (1.7 Å) were important in this respect. These crystal structures show that Asp156 and His126 are the two catalytic residues which are involved in proton donation and abstraction, respectively; when the (S)-enantiomeric substrate is bound in the active site and vice versa when the (R)-enantiomeric substrate is bound. The tight geometry of the active site also shows that His126 and Asp156 are involved in stabilizing the transition state. These crystal structures show that in the active site of MCR, there is one binding pocket for the CoA part and there are two different binding pockets (R-pocket and S-pocket) connected by a hydrophobic methionine rich surface for binding the fatty acyl part of the substrate. After substrate binding, proton abstraction takes place which produces a planar intermediate. Then, donation of a proton to the other side of the planar intermediate changes the configuration at the chiral center. During the stereochemical interconversion of the two enantiomers, the acyl group moves between R-pocket and S-pocket by sliding over the hydrophobic surface connecting these two pockets.

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Series: Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
ISSN-E: 1796-220X
ISBN: 951-42-8091-1
ISBN Print: 951-42-8090-3
Issue: 460
Copyright information: © University of Oulu, 2006. This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.