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 (αβ)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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/3869 |
| Date | 16 August 2006 |
| Creators | Cummings, Jennifer Ann |
| Contributors | Raushel, Frank M. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 1337344 bytes, electronic, application/pdf, born digital |
Page generated in 0.0021 seconds