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PEDS Advance Access published online on June 8, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh050
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Received February 5, 2004
Revised May 14, 2004
Accepted May 25, 2004

Article

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

I. V. Demidyuk 1*, D. V. Romanova 1, E. A. Nosovskaya 1, G. G. Chestukhina 2, I. P. Kuranova 3, S. V. Kostrov 1

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

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


  Abstract

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 glutamyl endopeptidase 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 glutamyl endopeptidases is not a key element determining their primary substrate specificity.

Keywords: Charge recognition, glutamyl endopeptidase, site-specific mutagenesis, serine protease, substrate specificity


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E.V. Gasanov, I.V. Demidyuk, A.V. Shubin, V.I. Kozlovskiy, O.G. Leonova, and S.V. Kostrov
Hetero- and auto-activation of recombinant glutamyl endopeptidase from Bacillus intermedius
Protein Eng. Des. Sel., November 1, 2008; 21(11): 653 - 658.
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