title: Purification and characterization of a prolidase from Aureobacterium esteraromaticum.
abstract: An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.
authors.affiliation: Central Laboratory, Asahi Chemical Industry Co. Ltd., Shizuoka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Fujii
authors.forename: Y
authors.initials: Y
authors.lastname: Nagaoka
authors.forename: S
authors.initials: S
authors.lastname: Imamura
authors.forename: T
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D000595:Amino Acid Sequence; D004150:Dipeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008670:Metals; D004150:Dipeptidases; C022449:proline dipeptidase