Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase.
The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using L-alanyl-beta-naphthylamide as substrate Vmax of the reaction was shown to be pH independent over the range of 5.5-9.0, while Km exhibited a pKa of 7.7. This latter value corresponds to the pKa of the amino group of the substrate. Using X-Ala and X-Leu to examine the specificity of the P1 site it was found that Arg and Lys exhibit the highest affinity, followed by Met, Val, Leu, Trp, and Phe, which bind congruent to 5- to 20-fold less well. Although Km varied more than 20-fold within this series, Vmax showed considerably less variation. Significantly weaker binding was observed with a P1 Gly, Ala, Ser, or Pro with no binding detectable with a P1 Glu. The presence of a P'1 Leu compared to P'1 Ala results in an approximate 10-fold decrease in Km with little change in Vmax. The effect of varying P'1 residues was examined with the series Leu-X. In this case basic and hydrophobic amino acids, with the exception of Val, all exhibit nearly the same Km. The binding of Arg-Arg and Lys-Lys showed the same Km as obtained for Arg-Leu or Lys-Leu, respectively. When Leu-Ser-Phe was compared to Leu-Ser the P'2 residue led to a 100-fold decrease in Km and slightly less than a 5-fold increase in Vmax. In contrast the addition of a P'2 Met to Leu-Trp results in only a 3-fold decrease in Km and a 3-fold increase in Vmax. The results indicate a preference for a basic or hydrophobic residue in the P1 and P'1 sites and indicate subsite-subsite interactions which primarily affect binding.
Johnson
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GD
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Hersh
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LB
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D004151:Dipeptides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity
D004151:Dipeptides; D009842:Oligopeptides; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme