Chemical modification of aminopeptidase isolated from Pronase.
Chemical modification of aminopeptidase from pronase has revealed two important histidines in enzyme catalysis. In the absence of metal ions, modification of the readily-modified histidine (pKa 6.9 +/- 0.5) results in a drastic loss of activity, indicating that this residue is indispensible for enzyme activity. In the presence of CaCl2, the modified enzyme still retains approx. 60% of the activity, whereas modification of another histidine (pKa 7.7 +/- 0.2) leads to a dramatic loss of activity. In fact, the enzyme with the first histidine being modified is active only in the presence of metal ions. Moreover, modification of the second histidine is prevented by the presence of Ca(II). These results indicate that the second histidine is serving as a ligand for Ca(II) and the bound Ca(II) is directly involved in enzyme catalysis. The c.d. spectra of the modified and unmodified enzymes in the absence or presence of CaCl2 are all very similar, indicating that no gross conformational changes in protein occur upon modification or by the presence of Ca(II). Modification of both histidines is prevented by the presence of a competitive inhibitor, suggesting that they are located in the active centre. Modification of 11 amino groups, two tyrosines, or four arginines causes no appreciable inactivation of the enzyme, indicating that these residues are not directly involved in enzyme catalysis.
Yang
S H
SH
Department of Chemistry, National Taiwan University, Taipei, Republic of China.
Wu
C H
CH
Lin
W Y
WY
D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002384:Catalysis; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011402:Pronase / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D006639:Histidine; D001120:Arginine; D000626:Aminopeptidases; D011402:Pronase; D004047:Diethyl Pyrocarbonate; D002122:Calcium Chloride; D002118:Calcium