CaltechAUTHORS
  A Caltech Library Service

Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the Mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3-3

Zhang, Pinghui and Liu, Suxing and Shan, Shu-ou and Ji, Xinhua and Gilliland, Gary L. and Armstrong, Richard N. (1992) Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the Mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3-3. Biochemistry, 31 (42). pp. 10185-10193. ISSN 0006-2960. doi:10.1021/bi00157a005. https://resolver.caltech.edu/CaltechAUTHORS:20191002-154651520

Full text is not posted in this repository. Consult Related URLs below.

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20191002-154651520

Abstract

Exons 1 and 2 and exon 8 of the mu class GSH transferases from rat encode sequence-variable regions 1 and 4 of mu class isoenzymes, respectively. These two of four variable regions are located at the N- and C-termini of this isoenzyme class and impinge on the active site. In order to assess the influence of these variable regions on the catalytic diversity of the class mu isoenzymes, seven chimeric isoenzymes were constructed by transplantation of the variable regions of the sequence of the type 4 subunit into the corresponding regions of the type 3 subunit. The chimeric isoenzymes exhibit unique catalytic properties. Replacement of all, or part, of variable region 4 of the type 3 subunit with that of the type 4 subunit results in chimeric catalysts with higher turnover numbers in nucleophilic aromatic substitution reactions. Analysis of the crystal structure of isoenzyme 3-3 [Ji, X., Zhang, P., Armstrong, R. N., & Gilliland, G. L. (1992) Biochemistry (preceding paper in this issue)] suggests that interaction of the flexible C-terminal tail with the N-terminal domain helps limit the rate of product release from the active site of isoenzyme 3-3 in this type of reaction. Substitution of all, or part, of the sequence-variable region 1 of subunit 3 with that of subunit 4 results in chimeric isoenzymes that mimic the high stereoselectivity but not the catalytic efficiency of isoenzyme 4-4 toward a,/3-unsaturated ketones. Modular mutagenesis of the N-terminal variable region together with linear free energy relationships using para-substituted 4-phenyl-3-buten-2-ones and the crystal structure of isoenzyme 3-3 has allowed the rapid location of a mutation, V9I, which appears to be crucial in determining the stereoselectivity of the chimeras and, by implication, isoenzyme 4-4 toward phenylbutenones and arene oxides.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/bi00157a005DOIArticle
ORCID:
AuthorORCID
Shan, Shu-ou0000-0002-6526-1733
Additional Information:© 1992 American Chemical Society. Received May 26, 1992; Revised Manuscript Received July 22, 1992. This work was supported by a grant from the National Institutes of Health (GM 30910). The authors thank Dr. Cecil Pickett and Professor David Tu and their research groups for generously providing the original cDNA clones from which the expression vectors were constructed and William W. Johnson for help with the fluorescence titrations.
Funders:
Funding AgencyGrant Number
NIHGM 30910
Issue or Number:42
DOI:10.1021/bi00157a005
Record Number:CaltechAUTHORS:20191002-154651520
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20191002-154651520
Official Citation:Modular mutagenesis of exons 1, 2, and 8 of a glutathione S-transferase from the Mu class. Mechanistic and structural consequences for chimeras of isoenzyme 3-3. Pinghui Zhang, Suxing Liu, Shu Ou Shan, Xinhua Ji, Gary L. Gilliland, and Richard N. Armstrong. Biochemistry 1992 31 (42), 10185-10193. DOI: 10.1021/bi00157a005
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99034
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 Oct 2019 22:58
Last Modified:16 Nov 2021 17:43

Repository Staff Only: item control page