Morpeth F F, Massey V
Biochemistry. 1982 Mar 16;21(6):1313-7. doi: 10.1021/bi00535a032.
The reaction of oxidized D-lactate dehydrogenase with D-lactate and reduced D-lactate dehydrogenase with pyruvate and oxygen was studied in a stopped-flow spectrophotometer. Oxidized enzyme was reduced in a triphasic manner by D-lactate at a variety of pH values. The fastest phase is thought to represent charge-transfer complex formation and is equal to an increase in long wavelength (510-700 nm) absorbance. The two slower phases seem to be due to flavin reduction. At pH 6 the proportion of the flavin reduction which proceeds by either phase is dependent upon the concentration of D-lactate. The reaction between reduced enzyme and oxygen is a simple second-order reaction. However, the oxidation of reduced D-lactate dehydrogenase by pyruvate is biphasic at pH 6 and monophasic at pH 8. At pH 8 there is a rapid increase in absorbance above 510 nm which is again ascribed to formation of a charge-transfer complex. These results are taken to indicate that D-lactate dehydrogenase may exist in two active conformations. At high pH the enzyme operates mainly through only one form while at low pH both forms participate in catalysis.
在停流分光光度计中研究了氧化型D - 乳酸脱氢酶与D - 乳酸的反应以及还原型D - 乳酸脱氢酶与丙酮酸和氧气的反应。在多种pH值下，氧化型酶被D - 乳酸以三相方式还原。最快的相被认为代表电荷转移复合物的形成，等同于长波长（510 - 700 nm）吸光度的增加。两个较慢的相似乎是由于黄素还原。在pH 6时，通过任一相进行的黄素还原比例取决于D - 乳酸的浓度。还原型酶与氧气之间的反应是简单的二级反应。然而，在pH 6时，丙酮酸对还原型D - 乳酸脱氢酶的氧化是双相的，在pH 8时是单相的。在pH 8时，510 nm以上吸光度迅速增加，这同样归因于电荷转移复合物的形成。这些结果表明D - 乳酸脱氢酶可能以两种活性构象存在。在高pH时，酶主要仅通过一种形式起作用，而在低pH时两种形式都参与催化作用。
