KINETIC STUDIES OF ENZYME MECHANISMS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 蛋白质可以含有二硫键或金属中心，当在体外操作时，这些金属中心会随着时间的推移而氧化。 该项目的目的是合成一种比现有化合物更好的氧化剂或在目前无法获得的pH范围内更活跃的氧化剂。一种化合物必须既是良好的氧化剂又能溶于水。 在25 º C的水中酸催化水解期间，在甲酰胺的反应中心测量了多种同位素效应。 该机制涉及羰基氧的快速预平衡质子化，随后形成至少一个对称四面体中间体，其不明显地与溶剂交换其羰基氧（kh/kex = 55）。通过15 N NMR测定甲酰胺的pKa，发现约为-2.0。 甲酰基氢动力学同位素效应-表明过渡态是高度四面体的（Dkobs = 0.79）;羰基碳动力学同位素效应（13 kobs = 1.031）与这一结论一致。 小的离去氮动力学同位素效应（15 kobs = 1.0050）与C-N键断裂之前的一些步骤一致，作为速率决定。羰基氧动力学同位素效应（18 kobs = 0.996）指出水的攻击作为速率决定步骤。 在这些结果的基础上，提出了一种机制，其中亲核试剂与质子化甲酰胺分子的连接是速率决定的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteins can contain disulfide bonds or metal centers which can unfortunately oxidize over time when manipulated in vitro. The purpose of this project is to synthetize a compound that is a better oxidizer than those compounds currently available or an oxidizer that is more active in a pH range not now available. A compound must be both a good oxidizer and be soluble in water. Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 ¿C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one symmetrical tetrahedral intermediate, which does not appreciably exchange its carbonyl oxygen with the solvent (kh/kex = 55). The pKa for formamide was determined by 15N NMR and found to be about -2.0. The formyl hydrogen kinetic isotope effect- is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl carbon kinetic isotope effect (13kobs = 1.031) is in agreement with this conclusion. The small leaving nitrogen kinetic isotope effect (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl oxygen kinetic isotope effect (18kobs = 0.996) points to attack of water as the rate-determining step. On the basis of these results a mechanism is proposed in which attachment of the nucleophile to a protonated formamide molecule is rate-determining.