title: Identification through combinatorial random and rational mutagenesis of a substrate-interacting exosite in the gamma domain of streptokinase.
creator: Yadav, Suman
creator: Aneja, Rachna
creator: Kumar, Prakash
creator: Datt, Manish
creator: Sinha, Sonali
creator: Sahni, Girish
subject: QD Chemistry
description: 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.
publisher: ASBMB
date: 2011-02-25
type: Article
type: PeerReviewed
format: application/pdf
identifier: http://crdd.osdd.net/open/485/1/sahni11.pdf
relation: http://www.jbc.org/content/286/8/6458.full.pdf+html
identifier:   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     
relation: http://crdd.osdd.net/open/485/