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Characterization of allantoinase from Eschericia coli

by Cummings, Jennifer Ann

Abstract (Summary)
The purpose of this research was to characterize the Escherichia coli, E. coli, allantoinase enzyme. Allantoinase catalyses the conversion allantoin to allantoate via the hydrolysis of a cyclic amide bond and is coded for by the allB gene. The enzyme is a member of the amidohydrolase superfamily. Amidohydrolase superfamily enzymes have a common (??)8-barrel structure but catalyze the hydrolysis of many different substrates by a common mechanism. The structural characteristics and roles of divalent cations of enzymes in this superfamily will be discussed and related to previous work conducted on allantoinases. In this work, the metal dependence of allantoinase was initially studied by Mn, Co, Zn, Cd, and Ni-supplemented assays of enzyme of very low metal content. By changing the growth conditions under which the allB was overexpressed in E. coli, and the addition of Zn, Co or Mn to the culture, enzyme with bound Zn (ZnALN), Co (CoALN) or Mn (MnALN) was produced. The pH dependence of log (kcat/KM) for allantoinase in the presence of MnCl2, ZnALN and CoALN followed a bell-shaped curve, indicating that one ionizable group needed to be deprotonated and the deprotonation of a second group caused a decrease in catalytic activity. The pK1 for ionization at low pH was dependent upon which divalent cation was present and is concluded to be that of the deprotonation of water. A structural model of allantoinase with bound allantoin was constructed and used to determine which amino acid residues may be involved in catalysis. Allantoinase mutants R67K, C152A, C152S, C287A, C287S, S317A, D315N and W332F were purified. The kinetic parameters kcat, KM and kcat/KM of wild type and mutant allantoinases were compared. The possible roles of these amino acid residues in catalysis and substrate binding, and the results of the pH rate profiles are discussed. A catalytic mechanism for allantoinase is proposed.
Bibliographical Information:

Advisor:Raushel, Frank M.; Fitzpatrick, Paul F.; Pettigrew, Donald W.

School:Texas A&M University

School Location:USA - Texas

Source Type:Master's Thesis

Keywords:allantoinase eschericia coli

ISBN:

Date of Publication:05/01/2003

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