Clemson University TigerPrints All Dissertations Dissertations 1-2010 Molecular Mechanics Studies of Enzyme Evolutionary Mechanisms Manoj Singh Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Chemistry Commons Recommended Citation Singh, Manoj, "Molecular Mechanics Studies of Enzyme Evolutionary Mechanisms" (2010). All Dissertations. 680. https://tigerprints.clemson.edu/all_dissertations/680 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. ABSTRACT In the current dissertation, closely related studies to quantify the mechanism underlying enzyme evolution have been discussed. The HIV-1 protease and !-lactamase enzymes were used as model systems for these studies. These are well known enzymes that are associated with drug resistance and are associated with the pathogenic diseases, and therefore, developing molecular level understanding of drug resistance through these enzymes has fundamental as well as practical importance. In chapter 2, the relationship between errors in modeled protein structures and associated binding affinity predictions to small molecules is established. The results of this study are applicable in addressing a wide range of biological questions including enzyme evolutionary mechanisms. The next three chapters discuss different aspects of HIV-1 protease evolution. In chapter 3, the role of substrate binding in manipulating the catalytic activity of HIV-1 protease during evolution has been examined. The results suggest that HIV-1 protease can optimize its catalytic activity by manipulating its substrate binding affinity. This part of study also emphasizes the importance of considering the in-vivo environment while studying physical-chemical aspects of enzymatic evolution. In chapter 4, the role of dynamics as a constraint on the evolution of HIV-1 protease has been examined. Low frequency motions (dynamics) of an enzyme have been suggested to be critical for its function. It has been further suggested that any mutation that disrupts these low frequency motions may have an adverse affect on the catalytic function of the enzyme. In this part of study, the role of dynamics as a constraint on the evolution of HIV-1 protease has been examined by comparing experimental ii activity data for over 90 mutants of HIV-1 protease to correlated motion data obtained from molecular dynamics simulations of a Michaelis complex. The results of this study suggest that dynamics do not impose a significant constraint on the evolution of HIV-1 protease. In chapter 5, the role of fold stability as a constraint on the evolution of HIV-1 protease is examined. A significant tradeoff between evolvability and fold stability for HIV-1 protease was observed in our study. The results of this study suggest that fold stability imposes a significant constraint on the evolution of HIV-1 protease, and in future attempts to predict evolutionary outcomes (drug resistant mutations), fold stability should also be taken into consideration. In chapter 6, the evolution of cefotaximase activity within !-lactamase is described. !-lactamase is a bacterial enzyme that catalytically hydrolyzes the !-lactam antibiotic, and therefore inactivates these drugs. Five point mutations are, however, required in the gene of this enzyme in order to develop cefotaximase activity. In this part of our study, we have studied the effect of four drug resistant amino acid mutations [A42G, E104K, G238S, and M182T] on the structural properties and cefotaximase activity of !-lactamase. Along with the successful identification of evolutionary beneficial mutations, our analyses suggest structural rearrangement within active site as a possible mechanism for increasing the activity against cefotaxime. iii DEDICATION To my family. iv ACKNOWLEDGMENTS I would like to thank, first of all, my advisor Prof. Brian Dominy. He has not only given me opportunity to pursue my PhD in the area of my interest, but has also been a wonderful mentor throughout my studies. I remember knocking his door countless number of times and he was always available for discussion and advice. He has taught me much more than science. I would also like to thank my graduate committee members for providing important comments and suggestions that greatly improved this dissertation. I would like to acknowledge Prof. Steve Stuart for his various courses which helped understand computational chemistry during my graduate studies. I am also very thankful to Computing and Information Technology department at Clemson University for installing and maintaining Palmetto cluster. Without this wonderful resource, my studies would have been less exciting. Finally, I would like to thank my family and friends. Without their constant support and encouragement, this journey would have been much more difficult. Manoj Kumar Singh December, 2010 v TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................i ABSTRACT.....................................................................................................................ii DEDICATION................................................................................................................iv ACKNOWLEDGMENTS ...............................................................................................v LIST OF TABLES..........................................................................................................ix LIST OF FIGURES ........................................................................................................xi CHAPTER I. INTRODUCTION .........................................................................................1 II. THERMODYNAMIC RESOLUTION: HOW DO ERRORS IN MODELED PROTEIN STRUCTURES AFFECT BINDING AFFINITY PREDICTIONS?.....................................................................8 Abstract....................................................................................................8 Introduction..............................................................................................9 Methods..................................................................................................11 Results and Discussion ..........................................................................17 vi Table of Contents (Continued) Page Conclusions............................................................................................29 III. THE EVOLUTION OF CATALYTIC FUNCTION IN THE HIV-1 PROTEASE..............................................................................................30 Abstract..................................................................................................30 Introduction............................................................................................32 Theoretical Background of Protease Activity Calculation ....................36 Methods..................................................................................................42 Results and Discussion ..........................................................................50 Conclusions............................................................................................68 IV. ROLE OF DYNAMICS AS A CONSTRAINT ON THE EVOLUTION OF HIV-1 PROTEASE....................................................69 Abstract..................................................................................................69 Introduction............................................................................................71 Methods..................................................................................................75 Results and Discussion ..........................................................................81 Conclusions............................................................................................96 V. ROLE OF FOLD STABILITY AS A CONSTRAINT ON THE EVOLUTION OF HIV-1 PROTEASE....................................................97 Abstract..................................................................................................97 vii Table of Contents (Continued) Page Introduction............................................................................................98 Theoretical Background.......................................................................102 Results..................................................................................................106 Discussion............................................................................................117 Methods................................................................................................119 VI. THE EVOLUTION OF CEFOTAXIMASE ACTIVITY IN THE TEM !-LACTAMASE...........................................................................122 Abstract................................................................................................122 Introduction..........................................................................................124 Methods................................................................................................131 Results..................................................................................................140 Discussion............................................................................................154