Nicotinamide adenine dinucleotide binding and promotion of enzyme activity: model based on affinity labeling of 3.alpha.,20.beta.-hydroxysteroid dehydrogenase with a nucleoside

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Abstract
5''-[p-(Fluorosulfonyl)benzoyl]adenosine (FSA) was used to affinity-label the NADH binding region of [Streptomyces hydrogenans] 3.alpha.,20.beta.-hydroxysteroid dehydrogenase (3.alpha.,20.beta.-HSD) to further test the hypothesis [Sweet et Samant (1980)] that 3.alpha. and 20.beta. activities occur at the same active site. Incubation of 3.alpha.,20.beta.-HSD (0.45 .mu.M) with FSA (125 .mu.M) at pH 7.40 and 0.degree. C caused simultaneous loss of 3.alpha. and 20.beta. activities by a 1st-order kinetic process, with t1/2 = 300 min for both activities. Dinucleotides and adenosine mononucleotides which acted as competitive inhibitors protected 3.alpha.,20.beta.-HSD against inactivation by FSA in a concentration-dependent manner, in the order reduced nicotinamide dinucleotide phosphate > oxidized nicotinamide dinucleotide phosphate > adenosine diphosphate-ribose > adenosine diphosphate > (AMP) > adenosine. Oxidized and reduced nicotinamide mononucleotides (NMH and NMNH) and steroid substrates did not protect 3.alpha.,20.beta.-HSD against affinity labeling by FSA. Although NMN was not a competitive inhibitor of 3.alpha.,20.beta.-HSD, NMN with AMP and also AMP with NMNH produced positive cooperativity for competitive inhibition of 3.alpha.,20.beta.-HSD. The results from FSA affinity labeling of the cofactor region confirm that both 3.alpha. and 20.beta. activities share the same active site of 3.alpha.,20.beta.-HSD and suggest a model of cofactor binding and promotion of enzyme activity. The adenosine 5''-phosphate component anchors the NAD or NADH to an adenosine domain in the cofactor binding region. The nicotinamide nucleotide component then carries out the H-transfer reaction at a neighboring domain near the steroid binding region.

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