Biochemistry, Department of

 

Date of this Version

4-17-2013

Comments

A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Donald F. Becker. Lincoln, Nebraska: April, 2013

Copyright (c) 2013 Nikhilesh Sanyal

Abstract

In cell metabolism, substrate channeling is a phenomenon where the product of one reaction is transported to a second enzyme active site without equilibrating into bulk solvent. Chapter 1 reviews the rationale and evidence for substrate channeling with the specific example of proline metabolism. Oxidation of proline to glutamate is catalyzed in consecutive reactions by proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH). The intermediate Δ1-pyrroline-5-carboxylate reportedly tends to be labile and inhibitory towards several metabolic pathways.

One of the main objectives of this dissertation was to investigate substrate channeling between independent proline oxidative enzymes from Thermus thermophilus- TtPRODH and TtP5CDH. Chapter 2 establishes that TtPRODH and TtP5CDH are capable of interacting with a dissociation constant (KD) of 3.03 µM as demonstrated using Surface Plasmon Resonance (SPR). As observed in the present study, this interaction is possible only with a specific orientation of TtPRODH relative to TtP5CDH. A docking model of the two enzymes predicts an orientation of the active sites which is supportive of substrate channeling. Corroborating observations are made with kinetic studies. We observe that interference of TtPRODH-TtP5CDH complex by catalytically inactive mutants TtPRODH R288M/R289M and TtP5CDH C322A lead to significant decrease in glutamate formation. The results pave the way for testing substrate channeling in eukaryotic enzymes. In chapter 3, two novel eukaryotic enzymes from Saccharomyces cerevisiae, Put1p (PRODH) and Put2p (P5CDH), have been characterized. Particular attention was focused on the oxidative half-reaction of Put1p for gaining insight into possible redox functions of human PRODH.

Previous studies show that bifunctional enzyme from Gram-negative Bradyrhizobium japonicum (BjPutA) containing PRODH and P5CDH domains, exhibits substrate channeling via an elegant internal tunnel. BjPutA and its channeling variants were used to test the role of substrate channel in hydrolysis of P5C, an essential step in proline oxidation. These aspects of substrate channeling are discussed in chapter 4.

Overall, this study provides an improved understanding of: (1) Substrate channeling in proline oxidation; and (2) a model for investigating substrate channeling between other individual enzymes that catalyze consecutive reactions.

Advisor: Donald F. Becker