Larry D. Sutton

Phenyl-n-butylborinic acid is a potent transition state analog inhibitor of lipolytic enzymes

The cholesterol esterase and lipoprotein lipase catalyzed hydrolyses of the water-soluble substrate p-nitrophenyl butyrate are competitively inhibited by butaneboronic acid and phenylboronic acid. Phenyl-n-butylborinic acid has been synthesized and characterized as an ultrapotent transition state analog inhibitor: Ki = 2.9 +/- 0.6 nM and 1.7 +/- 0.3 microM for the cholesterol esterase and lipoprotein lipase reactions, respectively. These results are interpreted in terms of transition state structure and stabilization.

Automation of polymerase chain reaction tests:Reduction of human errors leading to contamination

We compared the performance over 21 months of manually performed polymerase chain reaction (PCR)-based DNA analysis experiments with 25 months of automated PCR performed by a Zymark robotic system. Automation of the PCR technique resulted in a sixfold reduction in the number of experiments reporting carryover contamination and decreased the overall rate of contamination among total reactions 68-fold. Whereas contamination occurrences among manual experiments were evenly dispersed over the study interval and correlated with the lack of experience of laboratory personnel, the contamination that occurred with the robotic system was confined to the first 10 months of operation. In manual experiments, many of the 81 no-target false positives were sufficiently strong to result in the invalidation of 151 samples and positive controls. The seven no-target control false positives in the automated system were weak bands that were easily subtracted as background. Because none of the negative samples had DNA bands, no sample on the automated system has ever been invalidated as a result of contamination. Automation of PCR tests appears to offer great promise in reducing contamination to acceptable levels (e.g., < or = 0.1%).

Acylenzyme mechanism and solvent isotope effects for cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate

The mechanism of cholesterol esterase- (carboxylic ester hydrolase, EC 8.1.1.1) catalyzed hydrolysis of the water-soluble ester p-nitrophenyl butyrate has been characterized for commercially available preparations from bovine and porcine pancreas and for a purified preparation from porcine pancreas. Kinetic evidence for an acylenzyme mechanism is provided by experiments wherein the butyryl enzyme is trapped by MeOH, EtOH or n-BuOH. For the last alcohol the transacylation product n-butyl n-butyrate was characterized by GC-mass spectrometry. Solvent isotope effects have been measured for Vmax/Km, which is the rate constant for acylation, and for Vmax, which monitors rate-determining deacylation. Isotope effects of 1.5-3 on these rate constants indicate that both steps of the acylenzyme mechanism for cholesterol esterase catalysis involve transition states that are stabilized by general acid-base proton bridges.

Development, characterization, and initial evaluations of S1 a new chromogenic cephalosporin for β-lactamase delection

Abstract A novel, chromogenic cephalosporin reagent (S1) for β-lactamase testing was produced that shares physicochemical characteristics with nitrocefin (formerly 87/312). S1 and nitrocefin in a disk-testing format for β-lactamase performed at 100% agreement for detecting enzyme-producing isolates of Bacteroides fragilis group, Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, Staphylococcus aureus , and selected Enterobacteriaceae . The time required to achieve an initial color change or a strong positive reaction was comparable for both chromogenic reagents for all organisms except the Gram-positive species. S1 reaction times were ∼50% faster than nitrocefin for β-lactamase-positive enterococci and S. aureus . These results from the developmental studies and a commercially prepared disk lot indicate that S1 is a promising β-lactamase disk test reagent with the ability to detect all significant enzyme-producing species strains, some significantly earlier than the nitrocefin disk method.

Haloketone transition state analog inhibitors of cholesterol esterase

The cholesterol esterase-catalyzed hydrolysis of p-nitro-phenyl butyrate is reversibly inhibited by four phenyl haloalkyl ketones. Inhibitor potency is greatest for halogenated acetophenones and parallels the extent of hydration of the various ketones in buffered D2O. These results are consistent with an inhibition mechanism wherein haloketones reversibly form hemiketal adducts at the active site that structurally mimic tetrahedral intermediates of the cholesterol esterase catalytic cycle.

Dimensional mapping of the active site of cholesterol esterase with alkylboronic acid inhibitors

The cholesterol esterase-catalyzed hydrolysis of the water-soluble substrate p-nitrophenyl butyrate occurs via an acylenzyme mechanism, and is competitively inhibited by boronic acid transition state analog inhibitors. Accordingly, we undertook to dimensionally map the enzymes active site via synthesis and characterization of a series of n-alkyl boronic acid inhibitors. The most potent of these is n-hexaneboronic acid, with a Ki = 13 +/- 1 microM, since inhibitor potency declines for both longer and shorter boronic acids. No inhibition is observed for methaneboronic acid and n-octaneboronic acid inhibits poorly, with a Ki of 7 mM. These results indicate that the ability of the enzyme to form tight complexes with boron-containing transition state analog inhibitors is sensitive to alkyl chain length. The trend in inhibitor potency is discussed in terms of substrate specificity of and transition state stabilization by cholesterol esterase, and has important implications for the design of optimal reversible inhibitors of the enzyme.

Evaluation of S1 chromogenic cephalosporin β-lactamase disk assay tested against Gram-positive anaerobes, coagulase-negative staphylococci, Prevotella spp. and Enterococcus spp.

The efficacy of three rapid colorimetric disk assays to detect beta-lactamase production in 60 clinical isolates was evaluated. Two chromogenic cephalosporin substrates (S1 and nitrocefin) and an acidimetric test were in complete agreement when tested against Enterococcus spp. (20 strains, not Enterococcus faecalis), Prevotella spp. (10 strains) and Gram-positive anaerobic cocci (10 strains). However, the acidimetric test produced documented false-negative results in detecting the beta-lactamases from coagulase-negative staphylococci (two of 20 strains tested). The time required to produce a positive result for the discordant Staphylococcus epidermidis isolate favored S1 compared with nitrocefin. These studies indicate that the acidimetric test was less sensitive than the chromogenic cephalosporin substrates and that nitrocefin and S1 could be used to screen for beta-lactamase production in these tested species.

Prediction of piperacillin-tazobactam susceptibility among Enterobacteriaceae, pseudomonas aeruginosa, and other bacteria using ticarcillin-clavulanic acid, ceftazidime, and other broad-spectrum antimicrobial in vitro test results

The ability of various in vitro beta-lactam susceptibility test results to predict the susceptibility of piperacillin-tazobactam (a new beta-lactam-beta-lactamase inhibitor combination) was assessed using more than 46,000 recent clinical isolates. The organisms were tested by reference-quality National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution procedures and interpreted by the currently published NCCLS criteria. The recommended antimicrobial tests that would accurately predict the piperacillin-tazobactam in vitro efficacy had an overall very major, false-susceptible rate of only 0.6% (< or = 1.5% is acceptable). The following drug tests can be used to judge piperacillin-tazobactam activity and spectrum (low patient risk) conservatively: for Enterobacteriaceae use ticarcillin-clavulanic acid results (0.6% very major error); for Pseudomonas aeruginosa use piperacillin (0.1%) results; for enterococci use ampicillin or ampicillin-sulbactam (1.8%) results; for Haemophilus influenzae and Moraxella catarrhalis use cefotaxime or cefuroxime or ceftriaxone (1.5%); and for staphylococci use oxacillin by NCCLS recommendations. When the piperacillin-tazobactam testing reagents become available, the direct testing of this combination should be applied to relevant clinical isolates. The piperacillin-tazobactam break points should be reassessed as indicated by the cited minimum inhibitory concentration population analysis to improve predictive accuracy; H. influenzae susceptibility modified to < or = 2/4 micrograms/ml and Enterococcus species susceptibility tested at < or = 16/4 micrograms.

Phospholipase A1 activity and catalytic mechanism of pancreatic cholesterol esterase

Abstract The catalytic mechanism of bovine pancreatic cholesterol esterase (CEase) has been probed by using lipid p-nitrophenyl esters and thiophospholipids as substrates. The rate of CEase-catalyzed hydrolyses of p-nitrophenyl esters is highest for substrates that have fatty acyl chains of intermediate length, while solvent isotope effects decrease with increasing chain length. Nucleophilic trapping experiments indicate that kcat for these substrates is rate limited by hydrolysis of acylenzyme intermediates. The kcat for CEase-catalyzed hydrolysis of 1(3)-decanoylthio-2-decanoyl-phosphatidylcholine is nearly the same as that for p-nitrophenyl decanoate, which demonstrates that phospholipolysis is also rate limited by deacylation. Hence, the CEase and serine protease catalytic mechanisms are similar pro forma. This information is used to guide the design of mechanism-based inhibitors, two classes of which, phosphates and enolphosphates, are described herein.