SUICIDE SUBSTRATES FOR ALANINE RACEMASE OF ESCHERICHIA-COLI-B
SUICIDE SUBSTRATES FOR ALANINE RACEMASE OF ESCHERICHIA-COLI-B
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DOI：
10.1021/bi00600a028
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
WALSH, C
中科院分区：
文献类型：
--
作者：
WANG, E;WALSH, C
丙氨酸消旋酶（EC 5.1.1.1）来自E.大肠杆菌B纯化约9000倍，得到100，000道尔顿的二聚体，含有1分子磷酸吡哆醛/亚基。的不可逆的活性位点定向失活的酶的自杀底物的数量的分子基础进行了检查。β-的D和L异构体氟丙氨酸和β-氯丙氨酸在α，.β的丙酮酸、NH 3和卤素离子的消除或失活。未检测到外消旋化。从L-氯丙氨酸形成丙酮酸的Vmax比从氯丙氨酸或氟丙氨酸的D异构体形成的Vmax低约50倍。两种对映体对的分配相同，约830次失误/失活事件。这种不变的分配比表明，一个共同的中间体，eneamino酸-PLP [磷酸吡哆醛]复合物，是负责失活的物种，可能是由迈克尔攻击从亲核残基在酶的活性位点。与此想法一致，O-氨基甲酰基-D-丝氨酸和O-乙酰基-D-丝氨酸在引起不可逆失活之前也经历了830次酶催化消除，推测来自相同的中间体。O-氨基甲酰基-或O-乙酰丝氨酸的L异构体不消除也不诱导失活，而仅仅作为酶的可逆的竞争性抑制剂。这表明大体积β的不对称结合区域。在活性位点的取代基，并提出了取代的β-D异构体丙氨酸将优先是有效的酶灭活剂。D-环丝氨酸还以时间依赖性方式使丙氨酸消旋酶失活。因此，先前报道为可逆丙氨酸消旋酶抑制剂的天然抗生素O-氨基甲酰基-D-丝氨酸和D-环丝氨酸实际上与β-氨基甲酰基-D-丝氨酸一起沿着为自杀底物。卤代丙氨酸
Alanine racemase (EC 5.1.1.1) from E. coli B was purified about 9000-fold and a dimer of 100,000 daltons, containing 1 molecule of pyridoxal phosphate/subunit. The molecular basis for irreversible active site-directed inactivation of the enzyme by a number of suicide substrates was examined. Both the D and L isomers of .beta.-fluoroalanine and .beta.-chloroalanine partition between .alpha.,.beta. elimination to pyruvate, NH3 and halide ion or inactivation. No racemization is detectable. The Vmax for pyruvate formation from L-chloroalanine is approximately 50-fold lower than from the D isomer of chloroalanine or either fluoroalanine. Both enantiomeric pairs partition identically, approximately 830 turnovers/inactivating event. This invariant partition ratio suggests that a common intermediate, the eneamino acid-PLP [pyridoxal phosphate] complex, is the species responsible for inactivation, probably by Michael attack from a nucleophilic residue at the enzyme active site. In keeping with this idea, O-carbamoyl-D-serine and O-acetyl-D-serine also undergo enzyme-catalyzed elimination for 830 turnovers before causing irreversible inactivation, presumably from the same intermediate. The L isomers of O-carbamoyl- or O-acetylserine do not eliminate nor do they induce inactivation, but serve merely as reversible, competitive inhibitors of the enzyme. This suggests asymmetric binding regions for bulky .beta. substituents at the active site and suggests D isomers of substituted .beta.-alanines would be preferentially effective enzyme inactivators. D-Cycloserine also inactivates the alanine racemase in time-dependent fashion. Thus, both natural antibiotics O-carbamoyl-D-serine and D-cycloserine, previously reported as reversible alanine racemase inhibitors, are in fact suicide substrates along with the .beta.-haloalanines.