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PEDS Advance Access originally published online on June 8, 2004
Protein Engineering Design and Selection 2004 17(5):411-416; doi:10.1093/protein/gzh050
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Protein Engineering, Design & Selection vol. 17 no. 5 © Oxford University Press 2004; all rights reserved

Modification of substrate-binding site of glutamyl endopeptidase from Bacillus intermedius

I.V. Demidyuk1,4, D.V. Romanova1, E.A. Nosovskaya1, G.G. Chestukhina2, I.P. Kuranova3 and S.V. Kostrov1

1Laboratory of Protein Engineering, Institute of Molecular Genetics of Russian Academy of Sciences, Moscow 123182, 2Laboratory of Protein Chemistry, Institute of Genetics and Selection of Industrial Microorganisms, Moscow 113545 and 3Laboratory of Protein Crystallography, Institute of Crystallography of Russian Academy of Sciences, Moscow 117333, Russia

4 To whom correspondence should be addressed. E-mail: duk{at}img.ras.ru

Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family. Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear. The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system). In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213–Thr) and studied its catalytic properties. Such modification proved not to affect the primary specificity of the enzyme. The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times). The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity.

Received February 5, 2004; revised May 14, 2004; accepted May 25, 2004.

Edited by Robin Leatherbarrow


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E.V. Gasanov, I.V. Demidyuk, A.V. Shubin, V.I. Kozlovskiy, O.G. Leonova, and S.V. Kostrov
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