Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase

Gary D. Johnson, Louis B. Hersh

Research output: Contribution to journalArticlepeer-review

59 Scopus citations

Abstract

The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using l-alanyl-β-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 ∼-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.

Original languageEnglish
Pages (from-to)305-309
Number of pages5
JournalArchives of Biochemistry and Biophysics
Volume276
Issue number2
DOIs
StatePublished - Feb 1 1990

Bibliographical note

Funding Information:
’ This work was supported in part by National Institute on Drug Abuse Grant DA02243 and Welch Foundation Grant 1391. ’ TO whom correspondence should be addressed at Department of Biochemistry, University Texas Southwestern Medical Center, 5323 Harry Hines Blvd., TX 75235

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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