Direct NMR evidence that prolidase is specific for the trans isomer of imidodipeptide substrates.
The in vitro hydrolysis by porcine kidney prolidase of the imidodipeptide L-alanyl-L-proline was monitored by using 1H high-resolution NMR spectroscopy. The dipeptide exists as an equilibrium mixture of isomers with cis or trans conformation about the peptide bond. The 13C and 1H NMR spectra of the dipeptide displayed well-resolved resonances for each isomer. Inversion-transfer NMR spectroscopy, with a recently developed pulse sequence, was used with a range of temperatures to calculate the unitary rate constants for the exchange between isomers. A new analytical procedure was introduced for directly obtaining estimates of the unitary rate constants from inversion-transfer data. Arrhenius analysis yielded an activation energy for the isomerization of 87.0 +/- 4.1 kJ mol-1. 1H NMR time courses of the prolidase-catalyzed hydrolysis of L-alanyl-L-proline showed a faster removal of the trans isomer as the [enzyme]/[substrate] ratio was increased. The transient-kinetic information coupled with the steady-state kinetic parameters of the enzyme was used to develop two possible models of the overall hydrolytic reaction. Numerical integration of the relevant differential equations using the experimentally determined rate constants gave simulated progress curves that enabled selection of one of the proposed schemes as being the most likely; this proposal entailed absolute specificity of prolidase for the trans isomer of L-alanyl-L-proline. Finally, on the basis of the present work, and information from the literature, we have proposed a new model of the active site of the enzyme.
D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes; D004150:Dipeptidases / Q000378:metabolism*; D006859:Hydrogen; D007094:Imides; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine
D002247:Carbon Isotopes; D007094:Imides; D006859:Hydrogen; D004150:Dipeptidases; C022449:proline dipeptidase