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外文期刊>The biochemical journal
>Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain
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Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain
p1. 2-(N9-Acetyl-L-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2′-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2′-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N9-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites./p
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机译：> 1。合成了2-（N9-乙酰基-L-苯丙氨酰基）羟乙基2'-吡啶基二硫化物（化合物V），并研究了其与催化位反应的二级速率常数（k）的pH依赖性木瓜蛋白酶的硫醇基团（EC 3.4.22.2）用于评估在不存在P1-P2酰胺键的NH组分的情况下，存在疏水性占据位的酶S2亚位对于过渡态几何的后果。 2.化合物（V），2-（乙酰氨基）乙基2'-吡啶基二硫化物（化合物I）和2-（乙酰氧基）乙基2'-吡啶基二硫化物（化合物III）与K的pH依赖性的比较半胱氨酸蛋白酶最小催化位点模型苯并咪唑-2-基甲硫醇通过氢化作用建立了所有这些吡啶基二硫化物的活化作用，并且它们的反应性对分子非吡啶基部分的结构变化相对不敏感。因此，它们对木瓜蛋白酶反应性的显着差异源自直接或间接通过信号传导机制的结合。 3.比较木瓜蛋白酶与化合物（V）的反应动力学数据和与反应性探针进行类似反应的动力学数据，这些反应数据为S1-S2子位点之间的区域和S2子位点本身的各种结合相互作用提供了机会，从而导致得出以下结论：（a）木瓜蛋白酶与化合物（III）的（Gly-66）NH ... O = C小于（P1-P2酯）相互作用，相对于具有简单烃侧的探针而言，提供了更好的结合链，但没有信号传递给催化位点以提供（His-159）-ImH +辅助的过渡态; （b）当这种相互作用通过（P1-P2酰胺）NH ... O = C小于（Asp-158）相互作用（化合物I）或疏水性P2 / S2接触（化合物V）增强时，向发生催化区域以提供辅助过渡态; （c）当同时存在P2 / S2接触和涉及Gly-66的相互作用时，额外提供（P1-P2酰胺）NH ... O = C小于（Asp-158）相互作用[如2-（ N9-乙酰基-L-苯丙氨酰氨基）乙基2'-吡啶基二硫化物]仅用于协助结合，而没有额外的信号传导作用。 4.此类研究有望使仅将底物定位在适当酶基因座中的结合相互作用与那些将具有化学结果的信号传输至催化位点的结合相互作用区分开来。
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