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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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

Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (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 (D2O)k = 0.20 in 200 mM HCl, (D2O)k = 0.81 in 50 mM HCl, and (D2O)k = 4.2 in pure water. The formyl-H KIEs are (D)k = 0.80 in 200 mM HCl, (D)k = 0.77 in 50 mM HCl, (D)k = 0.75 in pure water, (D)k = 0.88 in 50 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 (18)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 (18)k = 0.9917 under neutral conditions, (18)k = 1.0290 (water nucleophile) or (18)k = 0.989 (hydroxide nucleophile) under alkaline conditions, and (18)(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.

Oxamate, an isosteric and isoelectronic inhibitory analogue of pyruvate, enhances the rate of enzymatic decarboxylation of oxaloacetate in the carboxyl transferase domain of pyruvate carboxylase (PC). It is unclear, though, how oxamate exerts a stimulatory effect on the enzymatic reaction. Herein, we report direct (13)C nuclear magnetic resonance (NMR) evidence that oxamate acts as a carboxyl acceptor, forming a carbamylated oxamate product and thereby accelerating the enzymatic decarboxylation reaction. (13)C NMR was used to monitor the PC-catalyzed formation of [4-(13)C]oxaloacetate and subsequent transfer of (13)CO(2) from oxaloacetate to oxamate. In the presence of oxamate, the apparent K(m) for oxaloacetate is artificially suppressed (from 15 to 4-5 μM). Interestingly, the steady-state kinetic analysis of the initial rates determined at varying concentrations of oxaloacetate and fixed concentrations of oxamate revealed initial velocity patterns inconsistent with a simple ping-pong-type mechanism. Rather, the patterns suggest the existence of an alternate decarboxylation pathway in which an unstable intermediate is formed. The steady-state kinetic analysis coupled with the normal (13)(V/K) kinetic isotope effect observed on C-4 of oxaloacetate [(13)(V/K) = 1.0117 ± 0.0005] indicates that the transfer of CO(2) from carboxybiotin to oxamate is the partially rate-limiting step of the enzymatic reaction. The catalytic mechanism proposed for the carboxylation of oxamate is similar to that proposed for the carboxylation of pyruvate, which occurs via the formation of an enol intermediate.

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)(NH(3))] 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)(NH(2)OH)] 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.

A kinetic investigation of the hydrolysis of semicarbazide by urease gives a relatively flat log V/ K versus pH plot between pH 5 and 8. A log V m versus pH plot shows a shift of the optimum V m toward lower pH when compared to urea. These results are explained in terms of the binding of the outer N of the NHNH 2 group in semicarbazide to an active site residue with a relatively low p K a ( approximately 6). Heavy-atom isotope effects for both leaving groups have been determined. For the NHNH 2 side, (15) k obs = 1.0045, whereas for the NH 2 side, (15) k obs = 1.0010. This is evidence that the NHNH 2 group leaves prior to the NH 2 group. Using previously published data from the urease-catalyzed hydrolysis of formamide, the commitment factors for semicarbazide and urea hydrolysis are estimated to be 2.7 and 1.2, respectively. The carbonyl-C isotope effect ( (13) k obs) equals 1.0357, which is consistent with the transition state occurring during either formation or breakdown of the tetrahedral intermediate.

Multiple kinetic isotope effects have been measured for the urease-catalyzed hydrolysis of formamide at pH 6.0 and 25 degrees C. These kinetic isotope effects include the carbonyl-C ((13)k = 1.0241 +/- 0.0009), the carbonyl-O ((18)k = 0.9960 +/- 0.0009), the formyl-H ((D)k = 0.95 +/- 0.01), the leaving-N ((15)k= 1.0327 +/- 0.0006), and the nucleophile-O ((18)k = 0.9778 +/- 0.0005). In addition, the enzyme does not catalyze the exchange of oxygen from the solvent into the carbonyl-O of formamide or the product, formate ion. The isotope effects are consistent with the rate-determining collapse of the tetrahedral intermediate (i.e., C-N bond cleavage). The pH optimum for formamide is at pH 5.3, whereas for urea, it is near 8.0. This is best accommodated by the mechanism proposed by Hausinger and Karplus, in which an active site cysteine binds to the nonleaving nitrogen in urea. For urea, the preference is for the anionic form of the sulfhydryl; for formamide, the neutral form is preferred, leading to the lower pH optimum.

Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 degrees C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one 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 (KIE) is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl-carbon KIE (13kobs = 1.031) is in agreement with this conclusion. The small leaving-nitrogen KIE (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl-oxygen KIE (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.

Multiple isotope effects have been measured for the acid-catalyzed hydrolysis of methyl formate in 0.5 M HCl at 20 degrees C. The isotope effects in the present investigation include the carbonyl carbon (13k = 1.028 +/- 0.001), the carbonyl oxygen (18k = 0.9945 +/- 0.0009), the nucleophile oxygen (18k = 0.995 +/- 0.001), and the formyl hydrogen ((D)k = 0.81 +/- 0.02). Determination of the carbonyl carbon, carbonyl oxygen, and formyl hydrogen isotope effects was performed via isotopic analysis of residual substrate. However, determination of the oxygen nucleophile isotope effect required analysis of the oxygen atoms of the product (formic acid), which exchange with the solvent (water) under acid conditions. This necessitated measurement of the rate of exchange of these oxygen atoms under the conditions for hydrolysis (k(ex) = 0.0723 min(-1)) and correction of the raw isotope ratios measured during the nucleophile-O isotope effect experiment. These results, along with the previously reported isotope effect for the leaving oxygen (18k = 1.0009) and the ratio of the rate of hydrolysis to that of exchange of the carbonyl oxygen with water (k(h)/k(ex) = 11.3), give a detailed picture of the transition-state structure for the reaction.

Using bioassays, the defensive behaviour of Crematogaster scutellaris and the biological activities of its abdominal secretion were investigated. Beside classical aggressive behaviours such as grips, C. scutellaris workers performed frequent characteristic gaster flexions during interspecific encounters, sometimes tempting to apply their abdominal secretion topically on the enemy. The toxicity of the venom of C. scutellaris to other ants greatly differed among the species tested, some being killed after the topical application of only three droplets, while others were quite resistant to a dose of 90 droplets. All ant species tested were strongly and immediately repelled by a contact between their antennae or mouthparts with the venom of C. scutellaris. Abdominal secretion was never used during intraspecific interference and workers were resistant to a topical application of the venom of their own species. Intraspecific repellency was significant but moderate compared to interspecific one. Workers of C. scutellaris were never seen using their venom during prey capture. In conclusion, the main biological activity of the abdominal secretion of C. scutellaris seems to be its repellency to other ant species. This is supported by field experiments showing that Pheidole pallidula foragers were efficiently repelled at coexploited baits, allowing the monopolization of most prey by C. scutellaris.

Acyl group transfer reactions, especially those to amides and esters, are important in biochemistry. Multiple kinetic isotope effects for the atoms at the reactive center of these molecules have provided the most detailed bonding picture of the transition state to date. These kinetic isotope effect studies are reviewed for several reactions of formamide, methyl benzoate, and methyl formate. In these cases all the evidence is consistent with a stepwise mechanism, involving tetrahedral intermediates. In the case of p-nitrophenyl acetate, the change to an excellent leaving group causes the tetrahedral intermediates to become kinetically unstable; the kinetic isotope effects are best fitted to a concerted mechanism.

The initial rates of incorporation of dTTP and thymidine 5'-O-(3-thiotriphosphate) (dTTP alpha S) into poly(dA) X oligo(dT) during template-directed synthesis by the large fragment of DNA polymerase I have been measured by using a rapid-quench technique. The rates were initially equal, indicating a nonrate-limiting chemical step. However, the rate of thionucleotide incorporation steadily diminished to 10% of its initial value as the number of consecutive dTMP alpha S residues in the primer strand increased. This anomalous behavior can be attributed to the helix instability inherent in phosphorothioate-containing duplexes. Positional isotope exchange experiments employing the labeled substrate [alpha-18O2]dATP have revealed negligible alpha, beta-bridging----beta-nonbridging isotope exchange in template-directed reactions of Escherichia coli DNA polymerase I (Pol I) both in the presence and in the absence of added inorganic pyrophosphate (PPi), suggesting rapid PPi release following the chemical step. These observations are consistent with a rate-limiting step that is tentatively assigned to a conformational change of the E X DNA X dNTP complex immediately preceding the chemical step. In addition, the substrate analogue (Sp)-dATP alpha S has been employed to examine the mechanism of the PPi exchange reaction catalyzed by Pol I. The net retention of configuration at the alpha-P is interpreted in terms of two consecutive inversion reactions, namely, 3'-hydroxyl attack, followed by PPi attack on the newly formed primer terminus. Kinetic analysis has revealed that while alpha-phosphorothioate substitution has no effect upon the initial rate of polymerization, it does attenuate the PPi exchange reaction by a factor of 15-18 fold.(ABSTRACT TRUNCATED AT 250 WORDS)

The SP diastereomer of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) is a substrate for the 32P-labeled inorganic phosphate exchange reaction catalyzed by the T and I forms of polynucleotide phosphorylase. The exchange reaction occurs with retention of configuration. This exchange reaction is very slow when only ADP alpha S(SP) is presented but is greatly activated by dinucleotide primers and ADP alpha S(RP), although the latter is not a substrate for the exchange reaction. Ap(S)A(RP) is an approximately 50% better activator of the exchange than the SP diastereomer. Furthermore, high levels of the ADP alpha S(SP) eliminate the activation by primers and by ADP alpha S(RP). A phosphatase activity is present with the I form of the enzyme which converts ADP alpha S(RP) to AMPS. This activity may be responsible for the formation of the 5'-phosphate end group for de novo polymerization or for the processivity of this reaction.

The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-phospho-poly(A). Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident. During de novo polymerization of the diastereomeric mixture, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete. The average chain length of the polymeric product is considerably smaller for ADP alpha S vs. ADP beta S or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.