Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 2. Reactions of the galactosyl-enzyme intermediate with alcohols and azide ion.
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 2. Reactions of the galactosyl-enzyme intermediate with alcohols and azide ion.
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β-半乳糖苷酶（大肠杆菌，lac Z）的结构-反应性关系。
DOI：
10.1021/bi00037a008
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Beard,J
中科院分区：
文献类型：
--
作者：
Richard,JP;Westerfeld,JG;Lin,S;Beard,J
1995年7月11日收到的修订版Mandarin pt ®摘要：通过将半乳糖基从β-半乳糖苷酶转移到七种烷基醇，葡萄糖，和叠氮离子被确定为β-半乳糖苷酶催化裂解4-硝基苯基β-D-吡喃半乳糖苷得到4-硝基酚氧阴离子（vpnp）的速度之差。通过该方法测定的半乳糖基化酶在与烷基醇和溶剂的反应之间的分配的速率常数比ε roh/s（M-1）与通过分析酶的醇抑制测定的β roh/s（M-1）的值良好一致。相应的烷基/3-D-吡喃半乳糖苷的催化水解。根据相应的速率常数比<$roh/^ s（M-1）和ks=710 s-1计算烷基醇与半乳糖基化酶中间体反应的绝对速率常数<$roh（M-1 s-1）。从醇与半乳糖基化酶反应的二级速率常数确定了Brpnsted参数（/3 μ c）roh=-0.19 ~0.10。烷基/3-D-吡喃半乳糖苷裂解形成半乳糖基化酶的（fig）kcur/Km=-0.75 ~0.14与逆合成反应的（f3 nUc）ROH=-0.19之间的巨大差异要求半乳糖基从烷基/3-D-吡喃半乳糖苷转移到酶的平衡常数随着烷基醇离去基团的pA”a的降低而急剧增加。对于烷基β-D-吡喃半乳糖苷与乙醇反应形成乙基β-D-吡喃半乳糖苷和烷基醇，这些数据给出β eq =-0.56到0.05。几种影响，导致这种增加的烷基/3-D-吡喃半乳糖苷的裂解的容易性与降低的烷氧基的碱度进行了讨论。测定了葡萄糖与半乳糖基化酶反应的二级速率常数kck= 1.2 × 104 M-1 s-1。葡萄糖的相对低的反应性是令人惊讶的，因为早期的观察表明，由乳糖裂解产生的半乳糖基化酶复合物以几乎相等的速率经历葡萄糖的释放和异乳糖的合成，这表明葡萄糖与半乳糖基化酶的结合应该是部分不可逆的，并且它发生在接近细菌控制的限度。数据表明，通过葡萄糖与半乳糖基化酶结合形成的非生产性复合物具有显著的稳定性。3-半乳糖苷酶催化β-D-吡喃半乳糖基叠氮化物的水解，但不催化通过叠氮离子与半乳糖基化酶的反应合成该化合物。这表明不同形式的β-半乳糖苷酶催化β-D-吡喃半乳糖基叠氮化物的裂解和合成。这可能对应于在裂解和合成方向上参与反应的酸碱催化的残基的电离状态的变化。3-半乳糖苷酶通过半乳糖基-酶反应中间体以两步机制催化乳糖和其他β-D-吡喃半乳糖衍生物的水解。Kulko-1.25的大次氘同位素效应（Sinnott和Souchard，1973）对来自大肠杆菌（lac Z）的酶的半乳糖基从酶转移到水的速率常数ks（s-1）的研究表明，在移动到中间体水解的过渡态时，在半乳糖基部分的碳-1处存在从sp3到sp2杂交的大变化，这需要中间体与酶的sp3-杂交共价连接。半乳糖基的共价连接点半乳糖基的共价连接点
Revised Manuscript Received July 11, 1995® abstract: Velocities for the synthesis of/3-D-galactopyranosyl derivatives by transfer of the galactosyl group from/3-galactosidase to seven alkyl alcohols, glucose, and azide ion have been determined as the difference in the velocities for/3-galactosidase-catalyzed cleavage of 4-nitrophenyl/3-D-galactopyranoside to give 4-nitrophenoxide anion (vpnp) and hydrolysis of this compound to give D-galactose Ocal)· Rate constant ratios£ roh/&s (M-1) for partitioning of the galactosylated enzyme between reaction with alkyl alcohols and solvent determined by this method are in good agreement with values of &roh/&s (M-1) determined by analysis of alcohol inhibition of enzyme-catalyzed hydrolysis of the corresponding alkyl/3-D-galactopyranosides. Absolute rate constants£ roh (M-1 s-1) for reaction of alkyl alcohols with the galactosylated enzyme intermediate were calculated from the corresponding rate constant ratio¿ roh/^ s (M-1) and ks=710 s-1. A Brpnsted parameter of (/3 „uc) roh=—0.19±0.10 was determined from the second-order rate constants for the reactions of alcohols with the galactosylated enzyme. The large difference between (fiig) kcur/Km=—0.75±0.14 for cleavage of alkyl/3-D-galactopyranosides to form the galactosylated enzyme and (/3nUc) ROH=—0.19 for the reverse synthesis reaction requires that the equilibrium constants for galactosyl group transfer from alkyl/3-D-galactopyranosides to theenzyme increase sharply with decreasing pA" a of the alkyl alcohol leaving group. These data give/3eq=-0.56±0.05 for the reaction of alkyl/3-D-galactopyranosides with ethanol to form ethyl/3-D-galactopyranoside and alkyl alcohol. Several effects that lead to this increased ease of cleavage of alkyl/3-D-galactopyranosides with decreasing basicity of the alkoxy group are discussed. A second-order rate constant of kck= 1.2 x 104 M-1 s-1 was determined for reaction of glucose with the galactosylated enzyme. The relatively low reactivity of glucose is surprising, because an earlier observation thatthe galactosylated enzyme complex generated by the cleavage of lactose undergoes release of glucose and synthesis of allolactose at nearly equal rates suggests that the binding of glucose to thegalactosylated enzyme should be partly irreversible and that it takes place near the encounter-controlled limit. The data suggest a significant stabilization of nonproductive complexes formed by binding of glucose to the galactosylated enzyme./3-Galactosidase catalyzes the hydrolysis of/3-D-galactopyranosyl azide, but not the synthesis of this compound by reaction of azide ion with the galactosylated enzyme. This suggests that different forms of/3-galactosidase catalyze the cleavage and synthesis of/3-D-galactopyranosyl azide. This may correspond to a change in the state of ionization of the residue that participates in acid-base catalysis of the reaction in the cleavage and synthesis directions./3-Galactosidase catalyzes the hydrolysis of lactose and other/3-D-galactopyranosyl derivatives by a two-step mech-anism through a galactosyl—enzyme reaction intermediate. The large secondary-deuterium isotope effect of kulko—1.25 (Sinnott & Souchard, 1973) for the enzyme from Escherichia coli (lac Z) on the rate constant ks (s-1) for transfer of the galactosyl group from enzymeto water shows that there is a large change from sp3 to sp2 hybridization at carbon-1 of the galactosyl moiety on moving to the transition state for hydrolysis of the intermediate, which requires sp3-hybridized covalent attachment of the intermediate to the enzyme. The point of covalentattachment of the galactosyl