Beta-secondary and solvent deuterium kinetic isotope effects on catalysis by the Streptomyces R61 DD-peptidase: comparisons with a structurally similar class C beta-lactamase.
Beta-secondary and solvent deuterium kinetic isotope effects on catalysis by the Streptomyces R61 DD-peptidase: comparisons with a structurally similar class C beta-lactamase.
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β-二级和溶剂氘动力学同位素对链霉菌 R61 DD-肽酶催化作用的影响：与结构相似的 C 类 β-内酰胺酶的比较。
DOI：
10.1021/bi982308x
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Pratt,RF
中科院分区：
文献类型：
--
作者：
Adediran,SA;Pratt,RF
已确定了链霉菌 R61 的 DD 肽酶和阴沟肠杆菌 P99 的 C 类 β-内酰胺酶对一系列无环底物周转的稳态动力学参数 V/K 和 V 的 β-二级和溶剂氘动力学同位素效应。尽管这些酶在进化上相关并且具有非常相似的三级和活性位点结构，但它们在功能上非常不同——前者有效地催化β-内酰胺的水解，但不能催化无环肽，而反之亦然适用于后者。测量的动力学同位素效应揭示了这些酶各种底物转换的稳态过渡态的相似性和差异。在大多数情况下，观察到逆β-次级同位素效应，反映了典型的酰基转移过渡态。然而，对于一种底物，间-[[(苯乙酰基)甘氨酰]氧基]苯甲酸，两种酶的同位素对V/K的影响非常接近一致。这些是根据酰化过渡态构象来解释的，其中 β-CH 超共轭的程度与游离底物中的程度相似。两种酶与该底物之间脱酰过渡态 (V) 的差异根据不同的酰基酶构象进行解释。对于两种酶和所有测试的底物，溶剂氘动力学同位素对 V/K 的影响均很小，有些甚至相反。从表面上看，这表明了一个违反直觉的结论：在所有这些情况下，酰化过渡态中几乎没有发生质子转移。然而，更仔细的分析表明，对于酯和酰胺（可能还有 β-内酰胺）底物，该结果可能是由于底物结合上质子分馏因子的增加被酰化过渡态的减少所抵消。前一个事件源自底物结合上的质子重排，而后者可能源自一般酸/碱催化。该结果对于所有β-内酰胺识别酶可能是通用的。溶剂同位素效应还表明，至少对于 P99 β-内酰胺酶，硫酯底物的酰化过渡态不涉及质子转移。这可以用四面体中间体的速率决定分解来解释，其中不需要离开的硫醇盐的质子化。两种酶的脱酰化过渡态似乎都涉及显着的质子转移，可能是由一般酸/碱催化引起的。
β-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parametersV/KandVfor turnover of a series of acyclic substrates by the DD-peptidase ofStreptomycesR61 and the class C β-lactamase ofEnterobacter cloacaeP99. Although these enzymes are evolutionarily related and have very similar tertiary and active site structure, they are functionally very differentthe former efficiently catalyzes the hydrolysis of β-lactams but not acyclic peptides while vice versa applies to the latter. The measured kinetic isotope effects reveal both similarities and differences in the steady-state transition states for turnover of the various substrates by these enzymes. In most cases, inverse β-secondary isotope effects were observed, reflecting typical acyl-transfer transition states. With one substrate, however,m-[[(phenylacetyl)glycyl]oxy]benzoic acid, isotope effects onV/Kof very close to unity were obtained for both enzymes. These were interpreted in terms of acylation transition state conformations where the extent of β-CH hyperconjugation was similar to that in the free substrate. Differences in deacylation transition states (V) between the two enzymes with this substrate were interpreted in terms of different acyl-enzyme conformations. Solvent deuterium kinetic isotope effects onV/Kwere uniformly small, some even inverse, for both enzymes and with all substrates tested. At face value, this suggests the counterintuitive conclusion that little proton transfer occurs in acylation transition states in all of these instances. Closer analysis, however, suggests that for ester and amide (and probably β-lactam) substrates, this result probably arises from an increase in proton fractionation factors on substrate binding being offset by their decrease in the acylation transition state. The former event derives from proton rearrangement on substrate binding and the latter, presumably, from general acid/base catalysis. This result may be general to all β-lactam-recognizing enzymes. The solvent isotope effects also suggest that, at least for the P99 β-lactamase, the acylation transition state of a thioester substrate does not involve proton transfer. This can be interpreted in terms of the rate-determining breakdown of a tetrahedral intermediate where no protonation of the leaving thiolate is required. Deacylation transition states of both enzymes appear to involve significant proton transfer, presumably arising from general acid/base catalysis.