Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck ...

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MUTATIONAL ANALYSIS OF AMINO ACID RESIDUES
INVOLVED IN ARGININOSUCCINATE LYASE ACTIVITY IN
DUCK 6 11 CRYSTALLIN
Anita Rupali Chakraborty
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Biochemistry
University of Toronto
@ Copyright by Anita Rupali Chakraborty (1998)
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ABSTRACT
MUTATIONAL ANALYSIS OF AMINO ACID RESIDUES
INVOLVED IN ARGININOSUCCINATE LYASE ACTIVITY IN
DUCK 6 I1 CRYSTALLIN
Anita Rupali Chakraborty
M.Sc. Thesis, 1998
Department of Biochemistry
University of Toronto
8 crystallins are the major structural eye lens protein of most birds and reptiles and are
direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isofoms of
6 crystallin, 8 I and 6 11, however only 6 II crystailin exhibits argininosuccinate lyase (ASL)
activity. At the onset of this study, the structure of argininosuccinate lyaseI6 II crystallin with
bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystalIographic
studies had suggested that H162 may function as the catalytic base in the argininosuccinate
lyase/6 [Icrystallin reaftion mechanism, either directly or through the activation of a water
molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate
substrate was modeled into the active site of duck 6 II crystallin, using the coordinates of an
inhibitor-bound E. coli fumaraseC structure to orient the fumarate moiety of the substrate. The
model served as a means of identifjmg active-site residues which are positioned to potentially
participate in substrate binding andlor catalysis. Based on the results of the modeling, site-directed
mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties
of each mutant determined. Kinetic studies reveal that five residues, R 11 5, N116, T 16 1, S283,
and E2% are essential for catalytic activity. Determination of the free energy of
unfolding/refolding of wild-type and mutant 6 II crystallins revealed that all constructs exhibit
similar thermodynamic stabilities. During the course of this work, the structure of an inactive
6 II crystallin mutant with bound substrate was solved (Valltk et al., 1998). This has allowed the
kinetic data to be interpreted on a structural basis and reveals that the kinetic and structural data are
in good agreement with one another.
I would like to thank the following people in recognition of their contribution to this thesis.
Firstly, my supervisor Dr. P. Lynne Howell, who has provided me with a perfect balance of
guidance and independence throughout my work.
My co-supervisors, Drs. A. Davidson and E. Pai for their valuable advice and suggestions
regarding my project.
Past and present members of the Howell laboratory, particularly Drs. Mary Turner and Fragois
Vallke, Liliana Sarnpaleanu, Chris Lemke, Patrick Yip, Gawen Thompson, and Mona Abu-Abed
for making the laboratory a very pleasant environment in which to work and providing valuable
technical expertise.
My parents Pushpa and Rathin N. Chakraborty and my brother Arnal for their encouragement and
patience throughout my studies.
Dr. Bill O'Brien for supplying the PET expression system for wild-type 6 II crystallin and Patrick
Yip for constructing the histidine-tagged expression system.
TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
1.1 The Lens Crystallins
1.2 The Ubiquitous Crystallins
1.2.1 a Crystallin
1.2.2 The ply Cry stallin Superfamily
1.2.2.1 f! Crystallins
1.2.2.2 ys Cry stallin
1L 2.3 y Crystallins
1.3 Gene Sharing and the Taxon-Specific Crystallins
1.3.1 6 Crystallin
1.4 Arghinosuccinate Ly ase (ASL)
1.4.1 Evidence of Gene Sharing
1.4.2 Function and Tissue Distribution
1.4.3 Gene Structure and Mutations
1.4.3.1 Argininosuccinic Aciduria
1.5 Catalytic Properties
1.5.1 The Catalytic Mechanism
1 S.2 Non-linear Enzyme Kinetics
1-53 Cold Dissociation
1 S.4 Inhibitors
1.5.5 The Elucidation of Active Site Residues
1.6 ASL Belongs to an Enzyme Superfamily
1.7 Thesis Objectives
1.8 Organization of the Thesis
CHAPTER 2: BACTERIAL EXPRESSION AND CHARACTERIZATION OF
WILD TYPE AND MUTANT DUCK 6 I1 CRYSTALLINS
2.1 Modeling the Substrate into the 6 JI Active Site 42
2.2 The Expression System 42
2.3 Site-Directed Mutagenesis 43
2.4 Bacterial Growth Conditions 45
2.5 Protein Purification 45
2.5.1 Cell Lysis 45
2.5.2 Cobalt Affmity Chromatography 46
2.5.3 Electrophoretic Characterization 46
2.5.4 Assay of Enzymatic Activity 47
2.6 Circular Dichroism Spectroscopy 50
2.6.1 Assessment of Secondary Structure 50
2.6.2 Reversible Denaturation of 6 I1 Crystallins in Guanidine Hydrochloride 50
CHAPTER 3: THERMODYNAMIC AND KINETIC PROPERTIES OF WILD
TYPE AND MUTANT S I1 CRYSTALLINS
3.1 Selection of Mutagenic Targets 5 1
3.2 Kinetic Characterization of 6 II Mutants 5 1
3.3 Assessing Conformational Integrity Using Circular Dichroism 53
3.3.1 Global Fitting of Reversible Guanidine Hydrochloride Denaturation 53
Using BIOEQS
CHAPTER 4: INTERPRETATION OF KINETIC AND THERMODYNAMIC
DATA
4.1 Assessment of the Modeling Studies 59
4.2 Circular Dichroism and Reversible Denaturation of 6 II Crystallins 62
4.2.1 Thermodynamic Stability of Wild-Type and Mutant 6 11 Cry stallins
4.2.2 Conformational Integrity of Wild-Type and Mutant 6 I1 Cry stallins
4.3 Roles of Active Site Residues in Catalysis
4.3.1 Residues Essential to Catalysis
4.3.1.1 The 280's Loop
4.3.1.2 The Charge-Relay System
4.4 Future Directions
4.4.1 Clarifying the Role of N116
4.4.2 Structures of Inhibitor-Bound 6 II Crystallin
BIBLIOGRAPHY