The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Specificity and immobilization of aminopeptidase.
We recently described the purification of two aminopeptidases from Streptomyces griseus (Vosbeck, K.D., Chow, K.-F., and Awad, W.M., Jr. (1973) J. Biol. Chem. 248, 6329-6034). An analysis of the amino acid composition reveals very little differences in the two proteins. Each protein has alanine as the NH2-terminal residue. The aminopeptidases were treated separately with acetic anhydride; as noted in the past, the presence of glycerol is required to achieve excellent yields of acetylated active derivatives (Siegel, S., and Awad, W.M., Jr. (1973 J. Biol. Chem. 248, 3233-3240). However, in the present case much higher concentrations of glycerol (50%) are needed during acetylation to obtain derivatives with completely reacted NH2-terminal residues. The epsilon-amino groups were not completely acetylated. In contrast to the native enzymes, the acetylated derivatives show an affinity for DEAE-cellulose, a property consonant with the changes in net charge. The kinetic constants for each enzyme against L-leucine-p-nitroanilide do not change significantly after acetylation. The specificities of the two aminopeptidases were examined extensively on a semiquantitative basis. The activities are not restricted by the length of substrate chains. Each enzyme shows a preference for hydrophobic residues at the ultimate and penultimate positions. Charge residues are released a slower rates. No prolidase activity is demonstrable even at high enzyme to substrate ratios; however, NH2-terminal proline residues are released readily. D-Amino acid residues at the ultimate or penultimate position substantially reduce the rate of hydrolysis; D-leucyl-D-leucine is not hydrolyzed...
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D003619:Dansyl Compounds; D005898:Glass; D005914:Globins; D006801:Humans; D007700:Kinetics; D011402:Pronase / Q000378:metabolism*; D011485:Protein Binding; D012995:Solubility; D013305:Streptomyces griseus / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D003619:Dansyl Compounds; D005914:Globins; D000626:Aminopeptidases; D011402:Pronase