Characterization of recombinant prolidase from Lactococcus lactis- changes in substrate specificity by metal cations, and allosteric behavior of the peptidase.
The Lactococcus lactis NRRL B-1821 prolidase gene was cloned and overexpressed in Escherichia coli. Under suboptimum growth conditions, recombinant soluble and active prolidase was produced; in contrast, inclusion bodies were formed under conditions preferred for cell growth. Recombinant prolidase retained more than half its full activity between 30 and 60 degrees C, and was completely inactivated after 30 min at 70 degrees C. CD analysis confirmed that prolidase was inactivated at 67 degrees C. The enzyme was active under weak alkali to weak acidic conditions, and showed maximum activity at pH 7.0. Although these characteristics are similar to those for other reported prolidases, this prolidase was distinctive for two kinetic characteristics. Firstly, different substrate specificity was observed for its two preferred metal cations, zinc and manganese: Leu-Pro was preferred with zinc, whereas Arg-Pro was preferred with manganese. Secondly, the enzyme showed an allosteric response to changes in substrate concentrations, with Hill constants of 1.53 for Leu-Pro and 1.57 for Arg-Pro. Molecular modeling of this prolidase suggests that these unique characteristics may be attributed to a loop structure near the active site.
Yang
Soo I
SI
Department of Food and Bioproduct Sciences, College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada.
Tanaka
Takuji
T
D000494:Allosteric Regulation; D001483:Base Sequence; D002412:Cations; D003001:Cloning, Molecular; D017931:DNA Primers; D004150:Dipeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D002412:Cations; D017931:DNA Primers; D011994:Recombinant Proteins; D004150:Dipeptidases; C022449:proline dipeptidase