Insights into the catalytic mechanism and active-site environment of Comamonas testosteroni delta 5-3-ketosteroid isomerase as revealed by site-directed mutagenesis of the catalytic base aspartate-38.
Delta 5-3-Ketosteroid isomerase (KSI) of Comamonas testosteroni catalyzes the isomerization of a wide variety of delta 5(6) and delta 5(10) steroids through the formation of an enzyme bound dienol(ate) intermediate. Asp-38 has been strongly implicated in catalysis, apparently serving as a proton shuttle. In this paper the results of a detailed kinetic characterization of the KSI mutants D38E and D38H are presented. Both mutants retain significant activity, with kcat and kcat/Km values 10(3)-10(4) times greater than the D38N mutant. The results allow for a qualitative assessment of the sensitivity of the enzymes catalytic capability to the positioning and chemical nature of the catalytic base. The near identity of the ratios of kcat5-AND/kcat5,10-EST is most easily explained by a mechanism in which the second chemical step, reketonization of the intermediate dienol(ate), is not significantly rate determining. The pH dependence of the rate constants for the D38E and D38H mutants is found to be consistent with earlier proposals that an as yet unidentified titrating functional group is present in the active site and indicates that the electrostatic environment of residue 38 is hydrophobic and positively charged.
Holman
C M
CM
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616, USA.
Benisek
W F
WF
D000735:Androstenedione / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet; D013251:Steroid Isomerases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D001224:Aspartic Acid; D000735:Androstenedione; C003528:5-androstene-3,17-dione; D013251:Steroid Isomerases; C022660:steroid delta-isomerase