KINETIC STUDIES OF ENZYME MECHANISMS

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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.