Tolosa E A, Maslova R N, Goryachenkova E V, Willhardt I H, Braunstein A E
Eur J Biochem. 1975 May 6;53(2):429-36. doi: 10.1111/j.1432-1033.1975.tb04083.x.
Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.
鸡肝中的丝氨酸巯基酶和鸡胚卵黄囊中的半胱氨酸裂解酶催化氨基酸底物的α - H原子与3 - H - 2O的交换。未反应底物的标记程度接近每摩尔氨基酸最多一个原子。在没有替代剂的情况下，底物中几乎没有H交换。积累的β - 取代产物的α - H完全被溶剂水的标记氢取代，与孵育时间无关。在3.5小时的孵育过程中，掺入过量（未反应）氨基酸底物中的标记α - 氢的量略少于掺入完全酶促β - 取代反应产物中的量。在检测灵敏度范围内，这些酶不会诱导氨基酸底物中β - H原子的同位素交换，对于L - 丝氨酸，也不会诱导18 - O标记的β - HO基团的同位素交换。丝氨酸巯基酶和半胱氨酸裂解酶都不会催化其底物的紧密结构类似物（如L - 丙氨酸、D - 丝氨酸、苏氨酸、3 - 磷酸丝氨酸）中的α - 氢交换。一个特殊情况是半胱氨酸裂解酶与竞争性抑制剂L - 丝氨酸（其抑制常数Ki等于L - 半胱氨酸的米氏常数Km）的相互作用：只有在存在共底物硫醇的情况下，裂解酶才以与L - 半胱氨酸大致相同的速率催化L - 丝氨酸中的α - H交换。目前关于底物氨基酸中同位素α - H交换的数据以及早期发表的证据表明，特异性β - 取代裂解酶的催化机制可能与消除或兼性（多功能）裂解酶的催化机制有显著不同。在特异性催化β - 取代反应的裂解酶情况下，形成α,β - 不饱和吡哆醛亚胺作为真正的反应中间体不太可能；这些反应可能通过本文提出的某种替代机制进行。
