[Frontiers in Bioscience 3, a23-26, May 1, 1998]

	[Frontiers in Bioscience 3, a23-26, May 1, 1998] Reprints PubMed CAVEAT LECTOR
	AMINOALKYLAZIRIDINES AS SUBSTRATES AND INHIBITORS OF LYSYL OXIDASE: SPECIFIC INACTIVATION OF THE ENZYME BY N-(5-AMINOPENTYL)AZIRIDINE Narasimhan Nagan¹, Patrick S. Callery² and Herbert M. Kagan¹ 1 Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, and the ² Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506 Received 4/2/98 Accepted4/6/98 5. DISCUSSION In summary, the inhibition of lysyl oxidase by APAZ is competitive, develops irreversibly with time, and is selective for the five-carbon chain length separating the amino and aziridinyl moieties. Since the compound is also productively and catalytically, although minimally, processed, in toto, the data indicate its interaction both as an inhibitor and as a substrate with the active site. Thus, it seems reasonable to speculate that APAZ initially interacts through its primary amino group with the LTQ cofactor, forming a Schiff base adduct with one of the ortho-carbonyls of this prosthetic group, following the usual course of amine oxidation (25). Since shorter and longer aminoalkylaziridines are substrates but do not inhibit the enzyme, this suggests that APAZ exists in its fully extended configuration upon such interaction with the active site, situating its electrophilic aziridine moiety proximal to an electron donating site of the enzyme. Moreover, since the pK_a (8.1) of free aziridine in aqueous solution approximates the pH (8.2) at which lysyl oxidase is assayed, it would be expected that approximately one-half of the available aziridinyl moieties would be protonated, thus increasing electrophilicity and generating a positive charge at that end of the molecule. Notably, oxyanions of dicarboxylic amino acid residues of other enzymes have been shown to covalently attack aziridine rings (12,14,15). Thus, it is of some interest that the sequence surrounding the carbonyl cofactor in rat lysyl oxidase is GCYDTYAADID, where the progenitor of the LTQ cofactor, tyr349, is represented in bold and italicized. Anionic charge stemming from the local abundance of aspartate residues may underlie the apparently exclusive preference of lysyl oxidase for a series of basic protein substrates whereas a variety of acidic proteins were not substrates for the enzyme (26). These aspartate residues are potential sources of nucleophilic oxyanions one of which may covalently react with the aziridine moiety of APAZ to inactivate lysyl oxidase. Indeed, molecular models illustrate the feasibility of a fully extended APAZ unit covalently bridging the b-carboxyl function of asp352 with the carbonyl cofactor, the latter linked through a Schiff base involving the primary amino function of APAZ. The future availability of isotopically labeled APAZ should permit the assessment of this hypothesis. The K_I and IC₅₀values for the inhibition by APAZ indicate that this reagent is not an unusually potent inhibitor of lysyl oxidase. Nevertheless, the results obtained with APAZ in the present study uniquely point to the potential of bifunctional reagents as covalent probes of the active site of this enzyme and should serve as a paradigm for the development of other inhibitors which might find use as anti-fibrotic agents.

[Frontiers in Bioscience 3, a23-26, May 1, 1998]
Reprints
PubMed
CAVEAT LECTOR

AMINOALKYLAZIRIDINES AS SUBSTRATES AND INHIBITORS OF LYSYL OXIDASE: SPECIFIC INACTIVATION OF THE ENZYME BY N-(5-AMINOPENTYL)AZIRIDINE

Narasimhan Nagan¹, Patrick S. Callery² and Herbert M. Kagan¹

1 Department of Biochemistry, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, and the ² Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506

Received 4/2/98 Accepted4/6/98

5. DISCUSSION

In summary, the inhibition of lysyl oxidase by APAZ is competitive, develops irreversibly with time, and is selective for the five-carbon chain length separating the amino and aziridinyl moieties. Since the compound is also productively and catalytically, although minimally, processed, in toto, the data indicate its interaction both as an inhibitor and as a substrate with the active site. Thus, it seems reasonable to speculate that APAZ initially interacts through its primary amino group with the LTQ cofactor, forming a Schiff base adduct with one of the ortho-carbonyls of this prosthetic group, following the usual course of amine oxidation (25). Since shorter and longer aminoalkylaziridines are substrates but do not inhibit the enzyme, this suggests that APAZ exists in its fully extended configuration upon such interaction with the active site, situating its electrophilic aziridine moiety proximal to an electron donating site of the enzyme. Moreover, since the pK_a (8.1) of free aziridine in aqueous solution approximates the pH (8.2) at which lysyl oxidase is assayed, it would be expected that approximately one-half of the available aziridinyl moieties would be protonated, thus increasing electrophilicity and generating a positive charge at that end of the molecule. Notably, oxyanions of dicarboxylic amino acid residues of other enzymes have been shown to covalently attack aziridine rings (12,14,15). Thus, it is of some interest that the sequence surrounding the carbonyl cofactor in rat lysyl oxidase is GCYDTYAADID, where the progenitor of the LTQ cofactor, tyr349, is represented in bold and italicized. Anionic charge stemming from the local abundance of aspartate residues may underlie the apparently exclusive preference of lysyl oxidase for a series of basic protein substrates whereas a variety of acidic proteins were not substrates for the enzyme (26). These aspartate residues are potential sources of nucleophilic oxyanions one of which may covalently react with the aziridine moiety of APAZ to inactivate lysyl oxidase. Indeed, molecular models illustrate the feasibility of a fully extended APAZ unit covalently bridging the b-carboxyl function of asp352 with the carbonyl cofactor, the latter linked through a Schiff base involving the primary amino function of APAZ. The future availability of isotopically labeled APAZ should permit the assessment of this hypothesis.

The K_I and IC₅₀values for the inhibition by APAZ indicate that this reagent is not an unusually potent inhibitor of lysyl oxidase. Nevertheless, the results obtained with APAZ in the present study uniquely point to the potential of bifunctional reagents as covalent probes of the active site of this enzyme and should serve as a paradigm for the development of other inhibitors which might find use as anti-fibrotic agents.