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The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of β-cyclodextrin (β-CD) on the surface of ZnS quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT: its linear range is from 0.1 ng mL -1 to 70 ng mL -1 , and the limit of detection can reach 0.03 ng mL -1 . The spiking recoveries of PCT in human serum samples range from 98.3% to 107%, with relative standard deviations ranging from 2.14% to 12.0%. This approach was applied to detect PCT in real patient serum samples, and the results are consistent with those obtained with a commercial ELISA kit.

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.

The membrane-bound beta-lactamase from Bacillus cereus, strain 569/H/9, has been purified to apparent homogeneity. Nonionic detergent (0.5% Triton X-100) is required to keep the enzyme (traditionally called gamma-penicillinase and now called beta-lactamase III) in solution. Antibodies to beta-lactamase III have been prepared, and the membrane-bound enzyme is immunochemically distinct from the extracellular enzymes. beta-Lactamase III has a molecular weight of 31 500, in contrast to the extracellular enzymes beta-lactamase I and beta-lactamase II which have molecular weights of 30 000 and 22 000, respectively. The isoelectric point of beta-lactamase III is pH 6.8, whereas beta-lactamase I and beta-lactamase II have isoelectric points about 8.6 and 8.3. The amino acid composition of beta-lactamase III differs from those of beta-lactamase I and beta-lactamase II; however, the difference index between the compositions of beta-lactamase I and beta-lactamase III (52%) suggests relatedness. beta-Lactamase III is inactivated by 6 beta-bromopenicillanic acid and by the sulfone of 6 alpha-chloropenicillanic acid, and cephalosporins are poorer substrates than penicillins. beta-Lactamase III may be a membrane-bound class A beta-lactamase.

The effect of replacement of sulfur in the cephem nucleus by oxygen upon the beta-lactamase stability, infrared carbonyl frequency of the beta-lactam ring, and antibacterial activity was investigated. The replacement reduced the stability of beta-lactam compounds to beta-lactamases, increased the IR frequencies, and enhanced the intrinsic antibacterial activity against bacterial strains without beta-lactamase. The instability of 1-oxacephalosporins to beta-lactamases, in other words, high reactivity to the enzymes, seemed to be due to the enhanced chemical reactivity of their beta-lactam rings which was indicated by their higher IR beta-lactam carbonyl frequencies. Based on a view that acylation of the enzyme by beta-lactam compounds occurred in both cases of beta-lactamase hydrolysis and target enzyme inhibition, the suggestion was made that one of the factors which conferred the higher intrinsic antibacterial activity on 1-oxacephalosporins was their high reactivity to the target enzyme(s), as was the case with beta-lactamases.

Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin. The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C. Three metallointermediates have been characterized by their visible absorption spectra. Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent [Bicknell, R., & Waley, S.G. (1985) Biochemistry 24, 6876-6887]. In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text). A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period. The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures. Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies. The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The cryoenzymology of several different beta-lactamases has been investigated. Particular attention has been paid to the experimental pitfalls of the technique. These include such factors as false bursts at the start of the reaction, instability of the enzymes during turnover, and Km values so high that little of the enzyme is present as a complex. Many of the difficulties in cryoenzymology stem from the use of organic cryosolvents. A novel "salt" cryosolvent has been tested: ammonium acetate solutions can be used down to about -60 degrees C. The enzymes examined are readily soluble, and stable, in this solvent. Nevertheless, out of 17 beta-lactamase beta-lactam systems, only 4 proved suitable for detailed investigation. In two of these, the hydrolysis of nitrocefin or 7-(thienyl-2-acetamido)-3-[[2-[[4- (dimethylamino)phenyl]azo]pyridinio]-methyl]cephem-4-carboxylic acid (PADAC), by beta-lactamase I from Bacillus cereus, substrate was converted into product at a slow enough rate (at -60 or -55 degrees C, respectively) for it to be possible to do successive scans during the course of the reaction. The spectra were those of substrate and product, and no intermediate was detected. The results argue against the accumulation of intermediate acyl-enzyme. The hydrolysis of PADAC by the P99 beta-lactamase from Enterobacter cloacae again showed spectra characteristic of substrate and product, and there was, moreover, a break in the Arrhenius plot; it is possible that a conformational change is (at least partially) rate-determining. The hydrolysis of dinitrophenylpenicillin by the P99 beta-lactamase did show features suggesting the accumulation of acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)