Avtor/Urednik		Kristan, Katja; Adamski, Jerzy; Lanišnik-Rižner, Tea; Stojan, Jure
Naslov		His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase
Tip		članek
Vir		Biochimie
Vol. in št.		Letnik 89, št. 1
Leto izdaje		2007
Obseg		str. 63-71
Jezik		eng
Abstrakt		17ß-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17(3-HSDcI) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of l7(3-HSDcI (Tyr167Phe, His164Trp/Gly, Tyr2l2Ala). Wild-type 17ß-HSDeI and the 17ß-HSDcI mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (VlK) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212A1a mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17(3-HSDcI have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.
Deskriptorji		17-HYDROXYSTEROID DEHYDROGENASES BINDING SITES MUTAGENESIS, SITE-DIRECTED KINETICS THERMODYNAMICS ASCOMYCETES