Active site-directed photoinactivation of delta 5-3-ketosteroid isomerase from Pseudomonas putida dependent on 1,4,6-androstatrien-3-one-17 beta-ol.
delta 5-3-Ketosteroid isomerase from Pseudomonas putida is subject to photoinactivation by light of wavelengths greater than 300 nm, specifically in the presence of the competitive inhibitor, 1,4,6-androstatrien-3-one-17 beta-ol (TEO). In the absence of this steroid or in the presence of the nonchromophoric steroidal competitive inhibitor, deoxycholate, the enzyme activity is essentially unaffected by irradiation. Deoxycholate protects the enzyme from the TEO-dependent reaction to a degree which is predictable from the concentrations of deoxycholate and TEO and their respective competitive inhibition constants, thus demonstrating that the inititial velocity of the photoinactivation is dependent upon the fraction of enzyme active sites occupied by TEO. Cholate, which is not a competitive inhibitor, does not protect enzyme activity. Amino acid analyses of hydrochloric acid hydrolysates of the photoinactivated enzyme show no significant differences from that of the native enzyme. However, the fraction of initial enzyme activity remaining correlates quantitatively with the disappearance of one of the four thiol groups in each polypeptide chain of the enzyme. Enzyme irradiated under the same conditions in the absence of TEO does not lose thiol groups.
D000596:Amino Acids / Q000032:analysis; D000733:Androstatrienes; D007535:Isomerases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008027:Light; D011549:Pseudomonas / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D000733:Androstatrienes; C023582:1,4,6-androstatrien-3-one-17 beta-ol; D007535:Isomerases; D013251:Steroid Isomerases