Catalytic Mechanism of C–C Hydrolase MhpC from Escherichia coli: Kinetic Analysis of His263 and Ser110 Site-directed Mutants


Li, C., Montgomery, M.G., Mohammed, F., Li, J.-J., Wood, S.P. and Bugg, T.D.H. (2004) Catalytic Mechanism of C–C Hydrolase MhpC from Escherichia coli: Kinetic Analysis of His263 and Ser110 Site-directed Mutants. Journal of Molecular Biology, 346, (1), 241-251. (doi:10.1016/j.jmb.2004.11.032). (Submitted).

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Description/Abstract

C–C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase)
from Escherichia coli catalyses the hydrolytic C–C cleavage of the meta-ring
fission product on the phenylpropionic acid catabolic pathway. The crystal
structure of E. coli MhpC has revealed a number of active-site amino acid
residues that may participate in catalysis. Site-directed mutants of His263,
Ser110, His114, and Ser40 have been analysed using steady-state and
stopped-flow kinetics. Mutants H263A, S110A and S110G show 104-fold
reduced catalytic efficiency, but still retain catalytic activity for C–C
cleavage. Two distinct steps are observed by stopped-flow UV/Vis
spectrophotometry, corresponding to ketonisation and C–C cleavage:
H263A exhibits very slow ketonisation and C–C cleavage, whereas S110A
and S110G exhibit fast ketonisation, an intermediate phase, and slow C–C
cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling
out a catalytic role, but shows a fivefold-reduced KM for the natural
substrate, and an ability to process an aryl-containing substrate, implying a
role for His114 in positioning of the substrate. S40A shows only twofoldreduced
catalytic efficiency, but shows a very fast (500 sK1) interconversion
of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating
that the enzyme accepts the dienol form of the substrate. These data imply
that His263 is responsible for both ketonisation of the substrate and for
deprotonation of water for C–C cleavage, a novel catalytic role in a serine
hydrolase. Ser110 has an important but non-essential role in catalysis,
which appears not to be to act as a nucleophile. A catalytic mechanism is
proposed involving stabilisation of reactive intermediates and activation of
a nucleophilic water molecule by Ser110.

Item Type: Article
ISSNs: 0022-2836 (print)
Related URLs:
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: University Structure - Pre August 2011 > School of Biological Sciences
ePrint ID: 35610
Date Deposited: 22 May 2006
Last Modified: 27 Mar 2014 18:22
URI: http://eprints.soton.ac.uk/id/eprint/35610

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