Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B

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Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the AG(U)(H 2 O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the k(cat)/K-M value by about 8- to 96-fold, but also increased the K-D value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 Angstrom resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.
Publisher
OXFORD UNIV PRESS
Issue Date
2003
Language
English
Article Type
Article
Keywords

HYDROGEN-BOND NETWORK; DELTA(5)-3-KETOSTEROID ISOMERASE; CRYSTAL-STRUCTURE; 3-OXO-DELTA(5)-STEROID ISOMERASE; DELTA-5-3-KETOSTEROID ISOMERASE; 3-OXO-DELTA-5-STEROID ISOMERASE; EQUILIBRIUM DISSOCIATION; CATALYTIC MECHANISM; TYROSINE TRIAD; TESTOSTERONI

Citation

JOURNAL OF BIOCHEMISTRY, v.134, no.1, pp.101 - 110

ISSN
0021-924X
DOI
10.1093/jb/mvg117
URI
http://hdl.handle.net/10203/85619
Appears in Collection
BS-Journal Papers(저널논문)
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