Use of azidobestatin as a photoaffinity label to identify the active site peptide of leucine aminopeptidase

Abstract

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. They are found in most cells and tissues, and their activity has been implicated in myriad fundamental biochemical and physiological processes. Nevertheless, little is known about the structure of the aminopeptidase active sites. Beef lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. Bestatin, [(2S,3R)-(3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] is a nonhydrolyzable substrate analogue of a peptide, PheLeu, which is rapidly cleaved by blLAP. Bestatin incorporates elements of the putative tetrahedral intermediate, and this results in a greater than 10(5)-fold enhancement of binding relative to analogous peptides. Bestatin is the most tightly bound inhibitor of many aminopeptidases. Bestatin was successively converted to nitrobestatin, p-aminobestatin, [3H]-p-aminobestatin, and finally [3H]-p-azidobestatin (pAB). Like bestatin, pAB is a slow binding inhibitor of LAP (Ki*, the dissociation constant for the final complex, = approximately 4 x 10(-9); Ki, the dissociation constant for the initial collision complex, = approximately 10(-8). The t1/2 for binding of 2 x 10(-8) M and 8 x 10(-8) M bestatin are approximately 60 min and approximately 38 min, respectively. pAB, nitrobestatin, bestatin, and physiological peptides appear to bind in the same site, the first three with similar avidity. In the dark, pAB and bestatin protect low concentrations of the enzyme against inactivation upon extensive dialysis. The t1/2 for photoactivation of pAB is approximately 3 s. Irradiation of blLAP for such short periods of time resulted in insignificant change in activity. blLAP which was placed in 254-nm light in the presence of pAB was inactivated significantly. Treatment of photolabeled blLAP with trypsin produces only two peptides. Autoradiography and scintillation counting indicate that the active site is in the peptide which includes residues 138-487. Treatment of the same blLAP with hydroxylamine produces two different peptides, with the active site in the peptide 323-487. This indicates that the active site is in the carboxyl-terminal one-third of the protomer. It is likely that this photoaffinity label will be useful in identifying active sites in other aminopeptidases as well.

Isolation of bovine kidney leucine aminopeptidase cDNA: Comparison with the lens enzyme and tissue-specific expression of two mRNAs

“…Its metal ion activation is also similar to that observed for bovine lens LAP (Stirling et al, 1989). In lens LAP, the C-terminal region contains the active site (Taylor et al, 1992), and we hypothesize that the same pertains for the E. coli peptidase since all the residues which appear to be involved in zinc binding and catalysis and all residues (except Met-454) which comprise the putative substrate binding site in bovine LAP are conserved in E. coli xerB (Burley et al, 1990; T ay lor, 1993a). These data suggest that these enzymes, Epithelium and cortex of young and old bovine lens were separated, and total RNA was extracted and enriched for poly(A) RNA.…”

“…The isolation of bovine kidney LAP cDNA provided us with a means to establish the correct amino acid sequence for LAP and to proceed with structural (Burley et al, 1992;Taylor et al, 1992) and mechanistic (Taylor et al, 1992(Taylor et al, ,1993 studies of the protein. It also allowed us to begin to explore regulation of LAP expression in lens epithelial cells during different stages in culture; in lens tissue at different stages of differentiation, maturation, and development; and in kidney tissue.…”

“…Bovine lens leucine aminopeptidase (LAP)1 was one of the first proteases discovered (Smith & Hill, 1960), and at present, it is the best-studied aminopeptidase with respect to composition (Carpenter & Vahl, 1973;Hanson & Frohne, 1976;Cuypers et al, 1982), structure (Taylor et al, 1979(Taylor et al, , 1992Burley et al, 1990), and mechanism of action (Allen et al, 1983;Taylor et al, 1982bTaylor et al, , 1992Taylor et al, , 1993. Immunological (Taylor et al, 1984a,b), mechanistic (Hanson et al, 1967, and structural (Oettgen & Taylor, 1985) data indicate that within a species LAPs from different tissues are indistinguishable and that interspecies differences are small.…”

The mercapturic acid pathway

“…The crystal structure of blLAP in complex with bestatin (267, Figure 22) shows that the a-amino group of the inhibitor interacts with Zn2 and the 2-hydroxyl group binds to both Zn1 and Zn2, and that the Phe and Leu sidechains occupy the S 1 and S 1 0 hydrophobic pockets near the active site (Burley et al 1991(Burley et al , 1992Kim et al 1993;Kim and Lipscomb 1994). The photoaffinity label, p-azidobestatin is also a slow, tight binding inhibitor of blLAP (Taylor et al 1992).…”

X-ray crystallographic determination of the structure of bovine lens leucine aminopeptidase complexed with amastatin: Formulation of a catalytic mechanism featuring a gem-diolate transition state

“…The amino acid sequence of blLAP also shows an overall 31% identity of residues and a 52% identity of residues in the C-terminal domain to that of the pepA-encoded aminopeptidase from Escherichia coli and Salmonella typhimurium (Stirling et al, 1988(Stirling et al, ,1989. In blLAP, the C-terminal domain contains the active site (Burley et al, 1990(Burley et al, , 1991(Burley et al, , 1992Taylor et al, 1992). Furthermore, the residues involved in metal coordination and inhibitor binding in blLAP, as revealed by the structure of the blLAPbestatin complex, are conserved in the pepA-encoded aminopeptidase (Burley et al, 1992).…”