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The hydrolysis of amides, oxoesters and thioesters is an important reaction in both organic chemistry and biochemistry. Kinetic isotope effects (KIEs) are one of the most important physical organic methods for determining the most likely transition state structure and rate-determining step of these reaction mechanisms. This method induces a very small change in reaction rates, which, in turn, results in a minimum disturbance of the natural mechanism. KIE studies were carried out on both the non-enzymatic and the enzyme-catalyzed reactions in an effort to compare both types of mechanisms. In these studies the amides and esters of formic acid were chosen because this molecular structure allowed development of methodology to determine heavy-atom solvent (nucleophile) KIEs. This type of isotope effect is difficult to measure, but is rich in mechanistic information. Results of these investigations point to transition states with varying degrees of tetrahedral character that fit a classical stepwise mechanism. This article is part of a special issue entitled: Enzyme Transition States from Theory and Experiment. [ABSTRACT FROM AUTHOR]

Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholines-terase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are D1Ok = 0.20 in 200 mM HCl D2Ok = 0.81 in 50 mM HCl, and D2Ok - 4.2 in pure water. The formyl-H KIEs are Dk = 0.80 in 200 mM HC1, Dk = 0.77 in 50 mM HC1, Dfc = 0.75 in pure water, Dk = 0.88 in SO mM NaOH, and D(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be ' k = 0.9917 under neutral conditions, 18fc = 1.0290 (water nucleophile) or 18fc = 0.989 (hydroxide nucleophile) under alkaline conditions, and i8(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented. [ABSTRACT FROM AUTHOR]

The urease-catalyzed hydrolysis of hydroxyurea is known to exhibit biphasic kinetics, showing a rapid burst phase followed by a slow plateau phase. Kinetic isotope effects for both phases of this reaction were measured at pH 6.0 and 25 °C. The observed nitrogen isotope effects for the ammonia leaving group [15(V/K)NH3] were 1.0016 ± 0.0005 during the burst phase and 1.0019 ± 0.0007 during the plateau phase, while those for the hydroxylamine leaving group [15(V/K)NH2OH] were 1.0013 ± 0.0005 for the burst phase and 1.0022 ± 0.0003 for the plateau phase. These isotope effects are consistent with a rate-determining step that occurs prior to breaking either of the two possible C-N bonds. The observed carbonyl carbon isotope effects [13(V/K)] were 1.0135 ± 0.0003 during the burst phase and 1.0178 ± 0.0003 during the plateau phase. The similarity of the magnitude of the carbon isotope effects argues for formation of a common intermediate during both phases. [ABSTRACT FROM AUTHOR]

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are 34 k = 0.994 ± 0.004, 13 k = 1.0148 ± 0.0007, and 18 k = 0.999 ± 0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC. [ABSTRACT FROM AUTHOR]

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, 13kobs = 1.0312, 18kobs = 0.997, and 34kobs = 0.995; for neutral conditions, 13kobs = 1.022, 18kobs = 1.010, and 34kobs = 0.996; and for alkaline conditions, 13kobs = 1.0263, 18kobs = 0.992, and 34kobs = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure. [ABSTRACT FROM AUTHOR]