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Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic beta-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and D-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acylenzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s(-1) and 75 s(-1), respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5[(phenylacetyl) amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the k(cat) for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the k(cat) of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The beta-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of beta-lactam antibiotics were obtained.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on N-15-enriched and C-13/N-15-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem. 203,483-491] to the side-chain H-1, N-15 and C-13 resonances. From both 3D heteronuclear H-1-NOE H-1-C-13 and H-1-NOE H-1-N-15 multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the Calpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl. Acad. Sci. USA 89, 2499 - 2503).

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on N-15-enriched and C-13/N-15-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem. 203,483-491] to the side-chain H-1, N-15 and C-13 resonances. From both 3D heteronuclear H-1-NOE H-1-C-13 and H-1-NOE H-1-N-15 multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the Calpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl. Acad. Sci. USA 89, 2499 - 2503).

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with N-15 and C-13 to overcome spectral overlap. Complete assignments were obtained for the backbone H-1, N-15 and C-13 resonances, using three-dimensional heteronuclear H-1 NOE (H-N)-H-1-N-15 multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation (H-N)-H-1-N-15 multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on N-15-enriched HPr and an additional three-dimensional triple-resonance (HN-N-C)-H-1-N-15-C-13-alpha correlation spectroscopy (HNCA) experiment on C-13, N-15-enriched HPr. Many of the sequential backbone H-1 assignments, as derived from two-dimensional NMR studies [Klevit, R. E., Drobny, G. P. & Waygood, E. B. (1986) Biochemistry 25,7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with N-15 and C-13 to overcome spectral overlap. Complete assignments were obtained for the backbone H-1, N-15 and C-13 resonances, using three-dimensional heteronuclear H-1 NOE (H-N)-H-1-N-15 multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation (H-N)-H-1-N-15 multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on N-15-enriched HPr and an additional three-dimensional triple-resonance (HN-N-C)-H-1-N-15-C-13-alpha correlation spectroscopy (HNCA) experiment on C-13, N-15-enriched HPr. Many of the sequential backbone H-1 assignments, as derived from two-dimensional NMR studies [Klevit, R. E., Drobny, G. P. & Waygood, E. B. (1986) Biochemistry 25,7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.