Beta-secondary Isotope Effect Research Articles

Acid‐Catalyzed Cyclization of Terpenes Under Homogeneous and Heterogeneous Conditions as Probed Through Stereoisotopic Studies: A Concerted Process with Competing Preorganized Chair and Boat Transition States

Based on stereoisotopic studies and beta-secondary isotope effects, we propose that the acid-catalyzed cyclization of geranyl acetate proceeds through a concerted mechanism. Under heterogeneous conditions (zeolite Y confinement), a preorganized chairlike transition state predominates, whereas under homogeneous conditions the boat- and chairlike transition states are almost isoenergetic. For the case of farnesyl acetate, we propose that under homogeneous conditions a concerted dicyclization occurs with a preorganized boat-chair transition state competing with the chair-chair transition state. Under zeolite confinement conditions, the chair-chairlike dicyclization transition state is highly favorable. The preference of chairlike transition states within the cavities of zeolite Y is attributed to a transition state shape selectivity effect.

Isotope Effects and syn Selectivity in the Ene Reaction of Triazolinedione with Conjugated Enones: Aziridinium Imide or an Open Intermediate Mechanism?

The ene reaction of 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) with phorone-d(6) (5-d(6)) shows an inverse beta-secondary isotope effect and with mesityl oxide-d(3) (7-d(3)) proceeds with 93% syn selectivity. These results are consistent with a mechanism involving the formation of an aziridinium imide intermediate in the rate-determining step. An open biradical or dipolar intermediate is excluded by them.

Beta-secondary and solvent deuterium kinetic isotope effects on beta-lactamase catalysis.

Beta-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]-oxy]benzoic acid, and of a beta-lactam substrate, penicillanic acid, by three typical class A beta-lactamases and a class C beta-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the beta-secondary isotope effects has been explicitly considered. The inverse beta-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1 beta-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM beta-lactamase and Bacillus cereus beta-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A beta-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C beta-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured beta-secondary isotope effect of 1,002 +/- 0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a beta-lactam and a depsipeptide by a beta-lactamase. The V transition state for both substrates with the P99 beta-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99 beta-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.

A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction.

The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.

Isotope effects in the solvolysis of haloallenes

Behavior of tri- and disubstituted haloallenes under solvolytic conditions was studied. Alpha- and beta-secondary isotope effects were measured. Experimental data support a carbonium ion mechanism. (LK)