creators_name: Yadav, Suman
creators_name: Aneja, Rachna
creators_name: Kumar, Prakash
creators_name: Datt, Manish
creators_name: Sinha, Sonali
creators_name: Sahni, Girish
type: article
datestamp: 2011-12-08 18:56:21
lastmod: 2011-12-08 18:56:21
metadata_visibility: show
title: Identification through combinatorial random and rational mutagenesis of a substrate-interacting exosite in the gamma domain of streptokinase.
ispublished: pub
subjects: QD
full_text_status: restricted
keywords: Enzyme CatalysisEnzyme KineticsEnzyme MechanismsEnzyme TurnoverEnzymesExosites
note: Copyright of this article belongs to ASBMB
abstract: To identify new structure-function correlations in the γ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the γ domain and its adjoining region in the β domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the γ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the native-like high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the γ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the γ domain of streptokinase.
date: 2011-02-25
date_type: published
publication: The Journal of biological chemistry
volume: 286
number: 8
publisher: ASBMB
pagerange: 6458-69
refereed: TRUE
issn: 1083-351X
official_url: http://www.jbc.org/content/286/8/6458.full.pdf+html
related_url_url: http://www.jbc.org/content/286/8/6458.full.pdf+html
related_url_type: pub
citation:   Yadav, Suman and Aneja, Rachna and Kumar, Prakash and Datt, Manish and Sinha, Sonali and Sahni, Girish  (2011) Identification through combinatorial random and rational mutagenesis of a substrate-interacting exosite in the gamma domain of streptokinase.  The Journal of biological chemistry, 286 (8).  pp. 6458-69.  ISSN 1083-351X     
document_url: http://crdd.osdd.net/open/485/1/sahni11.pdf