20 Beta-hydroxysteroid dehydrogenase of neonatal pig testis: cofactor requirement and stereospecificity of hydrogen transfer from nicotinamide adenine dinucleotide phosphate, reduced form.
The cofactor requirement of purified 20 beta-hydroxysteroid dehydrogenase from cytosol fraction of neonatal pig testis, in the reduction of 17 alpha-hydroxyprogesterone was investigated. The enzyme required beta-nicotinamide adenine dinucleotide phosphate, reduced form (beta-NADPH) as the preferred cofactor, with an apparent Km value of 17 microM. Furthermore, alpha-nicotinamide adenine dinucleotide phosphate, reduced form (alpha-NADPH), beta-3'-NADPH and beta-nicotinamide adenine dinucleotide (beta-NADH) were also utilized as hydrogen donors in the reduction at relatively high concentration with apparent Km values of 85.2 microM, 179.2 microM and 1.00 mM, respectively. The optimum pH was 5.5 when beta-NADPH was used as the cofactor, while it was 6.0 when beta-NADH was used. The hydrogen transfer from the beta-NADPH to the product, 17 alpha,20 beta-dihydroxypregn-4-en-3-one catalyzed by 20 beta-hydroxysteroid dehydrogenase was stereospecific, and the 4-pro-S-hydrogen of the nicotinamide moiety was transferred to the product.
Nakajin
S
S
Ohno
S
S
Aoki
M
M
Shinoda
M
M
D015089:20-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000818:Animals; D000831:Animals, Newborn; D006859:Hydrogen / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D013552:Swine; D013737:Testis / Q000201:enzymology*
D009249:NADP; D006859:Hydrogen; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase