Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping.
The stereochemistry of the L-histidinol dehydrogenase reaction was determined to be R at NAD for both steps, confirming previous results with a fungal extract [Davies, D., Teixeira, A., & Kenworthy, P. (1972) Biochem. J. 127, 335-343]. NMR analysis of monodeuteriohistidinols produced by histidinol/NADH exchange reactions arising via reversal of the alcohol oxidation reaction indicated a single stereochemistry at histidinol for that step. Comparison of vicinal coupling values of the exchange products with those of L-alaninol and a series of (S)-2-amino-1-alcohols allowed identification of the absolute stereochemistry of monodeuteriohistidinols and showed that histidinol dehydrogenase removes first the pro-S then the pro-R hydrogens of substrate histidinol. The enzyme stereochemistry was confirmed by isotope effects for monodeuteriohistidinols as substrates for the pro-R-specific dehydrogenation catalyzed by liver alcohol dehydrogenase. Active site mapping was undertaken to investigate substrate-protein interactions elsewhere in the histidinol binding site. Critical binding regions are the side-chain amino group and the imidazole ring, whose methylation at the 1- or 2-position caused severe decreases in binding affinity. Use of alternative substrates further clarified active site interactions with the substrate. Compounds in which the alpha-amino group was replaced by chloro, bromo, or hydrogen substituents were not substrates of the overall reaction at 1/10,000 the normal rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Grubmeyer
C T
CT
Department of Biology, New York University, New York 10003.
Insinga
S
S
Bhatia
M
M
Moazami
N
N
D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites; D006641:Histidinol / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
D006641:Histidinol; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase