Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles.
L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD. Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols. The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation. Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence. Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8. 4. In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.[1]References
- Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles. Grubmeyer, C., Teng, H. Biochemistry (1999) [Pubmed]
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