John F. Marlier

A highly efficient chemical synthesis of Rp and Sp adenyl(3′-5′)adenyl-o,o-phosphorothioate

Abstract A method is described for the chemical synthesis of the title compounds (Ap(S)A) via addition of elemental sulfur to a phosphite triester intermediate. Separation of the diastereomers of phosphorus could be accomplished on silica gel after removal of the terminal 5′-hydroxyl protecting group or on DEAE cellulose after complete deblocking. The triester to terminal 5′-hydroxyl. The overall yield of the synthesis (one coupling and four deblocking steps) is 30%.

Oxamate Is an Alternative Substrate for Pyruvate Carboxylase from Rhizobium etli

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.

A Kinetic and Isotope Effect Investigation of the Urease-Catalyzed Hydrolysis of Hydroxyurea

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 heavy-atom isotope effect and kinetic investigation of the hydrolysis of semicarbazide by urease from jack bean (Canavalia ensiformis).

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.

A kinetic isotope effect and isotope exchange study of the nonenzymatic and the equine serum butyrylcholinesterase-catalyzed thioester hydrolysis.

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.

A mechanistic study of thioester hydrolysis with heavy atom kinetic isotope effects.

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.

THE STEREOCHEMICAL COURSE OF SEVERAL ENZYME CATALYZED REACTIONS AT THE PHOSPHODIESTER LEVEL

Abstract A methodology is outlined for examining the stereochemical course at phosphorus of enzyme catalyzed reactions at the phosphodiester level utilizing phosphorothioates. Experiments with intestinal and venom phosphodiesterases that catalyze the formation of 5′-phosphonucleotides and poly-nucleotide phosphorylase that catalyzes the de novo polymerization of nucleotide diphosphates are reported. All of the reactions examined proceed with retention of configuration at phosphorus.