KINETIC-STUDIES OF AN ACTIVE-SITE DOUBLE MUTANT OF DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-PUTIDA BIOTYPE-B
SCIE
- Title
- KINETIC-STUDIES OF AN ACTIVE-SITE DOUBLE MUTANT OF DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-PUTIDA BIOTYPE-B
- Authors
- KIM, SW; CHOI, KY
- Date Issued
- 1995-08-31
- Publisher
- KOREAN SOC MOLECULAR BIOLOGY
- Abstract
- Delta(5)-3-Ketosteroid isomerase (KSI) promotes the highly efficient isomerization of Delta(5)-3-ketosteroids to Delta(4)-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position in order to compare the catalytic mechanisms of Pseudomonas putida isomerase with Comamonas testosteroni isomerase, well-conserved putative active-site amino acids in the P. putida KSI were replaced by site-directed mutagenesis: Asp-40 to asparagine and Tyr-16 to phenylalanine (D40N + Y16F). The double mutant isomerase was purified to homogeneity and characterized. The molecular weight of the purified double mutant was 14,517 (calculated, 14,519) as determined by electrospray mass spectrometry. While C. testosteroni D38N + Y14F double mutant showed no catalytic activity [Kuliopulos, A., Talalay, P., and Mirdvan, A. S. (1990) Biochemistry 29, 10271-10280], P. putida D40N + Y16F double mutant had some detectable activity. The k(cat) value of the double mutant enzyme is 10(6.6)-fold lower than that of the wild-type enzyme and K-m value is similar to that of wild-type enzyme. Although active-site residues of the two homologous enzymes are well conserved and of similar properties, kinetic properties of P. putida double mutant isomerase are not similar to those of the C. testosteroni double mutant. This result suggests that the precise mechanism of both homologous enzymes are somewhat different.
- Keywords
- DELTA-5-3-KETOSTEROID ISOMERASE; CATALYTIC MECHANISM; MUTATIONS
- URI
- https://oasis.postech.ac.kr/handle/2014.oak/21730
- ISSN
- 1016-8478
- Article Type
- Article
- Citation
- MOLECULES AND CELLS, vol. 5, no. 4, page. 354 - 358, 1995-08-31
- Files in This Item:
- There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.