Gondry M, Lederer F
URA 1461, Centre National de la Recherche Scientifique, Hôpital Necker, Paris, France.
Biochemistry. 1996 Jul 2;35(26):8587-94. doi: 10.1021/bi9529519.
The FMN prosthetic group of flavocytochrome b2 or L-lactate dehydrogenase oxidizes lactate to pyruvate. The reducing equivalents are then transferred one by one, intramolecularly, to heme b2 and then to external acceptors. Substrate oxidation is thought to begin with abstraction of the substrate alpha-hydrogen as a proton by an enzyme base. It has been proposed that this role is played by His373, which lies close to the flavin in the crystal structure and interacts with Asp282. It has also been shown before, using hydrogen exchange measurements, that the pKa of His373 is substantially increased in the wild-type reduced enzyme compared to that in the oxidized state. We report here the enzymatic properties of the D282N mutant flavocytochrome b2. Steady-state rate measurements with [2-1H]lactate and [2-2H]-lactate indicate that, as predicted, the Michaelis complex stability is hardly affected, whereas the transition state for proton abstraction increases in energy by 2.8 kcal/mol. Steady-state inhibition studies were conducted with a number of active-site ligands: sulfite, D-lactate, pyruvate, and oxalate. Binding was found to be most affected for oxalate, but kinetic patterns indicated oxalate and pyruvate were still capable of binding to the enzyme both at the oxidized and semiquinone stages, whereas inhibition by excess substrate, due to lactate binding at the semiquinone stage, was lost. Finally, analysis of the intermolecular hydrogen transfer catalyzed by the enzyme between [2-3H]lactate and fluoropyruvate indicated that the substitution with asparagine facilitates exchange of the histidine-bound proton and hence induces a decrease in the pKa value of H373 in the reduced enzyme of about 1.4 pH units. Nevertheless, the rate constant value for exchange with the solvent of the enzyme-bound substrate alpha-proton indicates that H373 is still protonated in the reduced mutant enzyme at neutral pH. Thus, the D282N mutation destabilizes the transition state for proton abstraction and decreases the pKa of H373 in the reduced enzyme but is insufficient to bring it back to a normal value.
黄素细胞色素b2或L-乳酸脱氢酶的黄素单核苷酸辅基将乳酸氧化为丙酮酸。然后，还原当量在分子内逐个转移至血红素b2，再转移至外部受体。底物氧化被认为始于底物的α-氢被酶碱作为质子提取。有人提出，这一作用由His373发挥，它在晶体结构中靠近黄素，并与Asp282相互作用。之前也已表明，通过氢交换测量，野生型还原酶中His373的pKa值与氧化态相比大幅增加。我们在此报告D282N突变型黄素细胞色素b2的酶学性质。用[2-1H]乳酸和[2-2H]乳酸进行的稳态速率测量表明，正如预测的那样，米氏复合物稳定性几乎未受影响，而质子提取的过渡态能量增加了2.8千卡/摩尔。用多种活性位点配体进行了稳态抑制研究：亚硫酸盐、D-乳酸、丙酮酸和草酸盐。发现草酸盐对结合的影响最大，但动力学模式表明草酸盐和丙酮酸在氧化态和半醌态时仍能与酶结合，而由于乳酸在半醌态结合导致的过量底物抑制作用消失。最后，对酶催化的[2-3H]乳酸和氟丙酮酸之间的分子间氢转移分析表明，用天冬酰胺取代有助于组氨酸结合质子的交换，从而导致还原酶中H373的pKa值降低约1.4个pH单位。然而，与酶结合底物α-质子的溶剂交换速率常数表明，在中性pH下，还原突变酶中的H373仍被质子化。因此，D282N突变使质子提取的过渡态不稳定，并降低了还原酶中H373的pKa值，但不足以使其恢复到正常值。
