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Crystal structure of Delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization

Title
Crystal structure of Delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization
Authors
Cho, HSHa, NCChoi, GKim, HJLee, DOh, KSKim, KSLee, WChoi, KYOh, BH
POSTECH Authors
Choi, KYOh, BH
Date Issued
Jan-1999
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLO
Abstract
Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization, Asp(38) (PKalpha similar to 4.7) was identified as the general base abstracting the steroid C4 beta proton (pK(alpha) similar to 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 Angstrom crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(1.4) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin, The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.
Keywords
CATALYZED PROTON ABSTRACTION; CARBON ACIDS; 3-OXO-DELTA(5)-STEROID ISOMERASE; DELTA-5-3-KETOSTEROID ISOMERASE; ENZYMATIC CATALYSIS; SERINE PROTEASES; INTERMEDIATE; MECHANISM; RESIDUES; D38N
URI
http://oasis.postech.ac.kr/handle/2014.oak/20217
ISSN
0021-9258
Article Type
Article
Citation
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 46, page. 32863 - 32868, 1999-01
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 CHOI, KWAN YONG
Div of Integrative Biosci & Biotech
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