Mechanism of Salmonella typhimurium histidinol dehydrogenase: Kinetic isotope effects and pH profiles

L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histid inol to L-histidine with sequential reduction of two molecules of NAD. Prev ious isotope exchange results had suggested that the oxidation of histidino l to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) s tudies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterat ed histidinols, The data at pH 9.0 support minimal participation of the fir st hydride transfer and substantial participation of the second hydride tra nsfer in the overall rate limitation. Stopped-flow experiments with protiat ed histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, ind icating that the overall first half-reaction was not significantly faster t han the second reaction sequence. Results from k(cat) and k(cat)/K-M titrat ions with histidinol, NAD, and the alternative substrate imidazolyl propane diol demonstrated an essential base with pK(a) values between 7.7 and 8.4. In KIE experiments performed at pH 5.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulation s based on rate constants estimated from this work fit well with a mechanis m that includes a relatively fast, and thermodynamically unfavorable, hydri de transfer from histidinol and a slower, irreversible second hydride trans fer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.