Hansen B A, Dekker E E
Biochemistry. 1976 Jun 29;15(13):2912-7. doi: 10.1021/bi00658a033.
Kinetic data show that the irreversible inactivation of liver 2-keto-4-hydroxyglutarate aldolase observed when the enzyme is incubated with an aldehydic substrate (or substrate analogue) in the presence of cyanide is a biphasic process and can, under certain conditions, involve a direct interaction between the enzyme and cyanide. The kinetic data are consistent with a scheme consisting of three competing reactions: (1) irreversible addition of cyanide to the enzyme-substrate Schiff base intermediate, (2) reversible cyanohydrin formation between cyanide and the aldehydic substrate (or substrate analogue), and (3) an interaction of cyanide with the enzyme which is not substrate dependent. Approximately 0.4 mol of cyanide is associated with 1 mol (120 000 g) of enzyme when 2-keto-4-hydroxyglutarate aldolase is incubated with [14-C]-cyanide followed by exhaustive dialysis; an ionic attachment possibly at a carboxylate binding site, is suggested. Whereas native enzyme, not treated with cyanide, has ten Nbs2-titratable sulfhydryl groups, approximately one less such group reacts with Nbs2 when the aldolase is incubated with cyanide (in the absence of aldehydic substrate). It is suggested that the binding of cyanide results in a conformational change of the enzyme; conformational changes in the presence of cyanide are confirmed by circular dichroism spectra.
动力学数据表明，当肝2-酮-4-羟基戊二酸醛缩酶在氰化物存在下与醛底物（或底物类似物）一起孵育时观察到的不可逆失活是一个双相过程，并且在某些条件下可能涉及酶与氰化物之间的直接相互作用。动力学数据与一个由三个竞争反应组成的机制一致：（1）氰化物不可逆地加成到酶-底物席夫碱中间体上，（2）氰化物与醛底物（或底物类似物）之间可逆地形成氰醇，以及（3）氰化物与不依赖底物的酶之间的相互作用。当2-酮-4-羟基戊二酸醛缩酶与[14-C]-氰化物一起孵育然后进行彻底透析时，约0.4摩尔的氰化物与1摩尔（120000克）的酶结合；提示可能是在羧酸盐结合位点的离子性结合。未用氰化物处理的天然酶有10个可被Nbs2滴定的巯基，而当醛缩酶与氰化物一起孵育（在无醛底物的情况下）时，与Nbs2反应的此类基团大约少一个。提示氰化物的结合导致酶的构象变化；圆二色光谱证实了在氰化物存在下的构象变化。
