Date of Award

1-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Chemistry

Advisor

Dominy, Brian N

Committee Member

Stuart , Steven J

Committee Member

McNeill , Jason

Committee Member

Alexov , Emil

Abstract

In the current dissertation, closely related studies to quantify the mechanism underlying enzyme evolution have been discussed. The HIV-1 protease and beta-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 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 beta-lactamase is described. beta-lactamase is a bacterial enzyme that catalytically hydrolyzes the beta-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 beta-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.

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