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
Received 4/2/98 Accepted4/6/98
TABLE OF CONTENTS
1. ABSTRACT
The reaction of lysyl oxidase was assessed with members of a series of aminoalkylaziridines in which the primary amino group and the aziridinyl nitrogen were separated by 3-7 methylene carbons. Among these, N-(5-aminopentyl)aziridine proved to be the poorest substrate by far and to inhibit the enzyme activity. Aminoalkylaziridines with chain lengths shorter or longer than five carbons did not inhibit the enzyme. The resulting inhibition was competitive with productive substrates and became irreversible with time, following pseudo first order kinetics with a KI of 0.22 mM. N-(5-aminopentyl)aziridine appears to act as a bifunctional affinity label covalently interacting with the active site of this enzyme.
2. INTRODUCTION
Lysyl oxidase is a copper-dependent amine oxidase which initiates the biosynthesis of lysine-derived crosslinkages by oxidizing peptidyl lysine residues to alpha-aminoadipic-delta-semialdehyde in elastin and collagen. This peptidyl aldehyde is the precursor of the variety of crosslinkages found in these proteins (1,2). In addition to copper, lysyl oxidase also contains a covalently bound carbonyl prosthetic group which is essential for catalytic function. This cofactor has recently been identified in rat lysyl oxidase as lysyl topaquinone (LTQ) deriving from tyrosine 349 and lysine 314 (3). The irreversible inactivation of this enzyme by 1,2-alkyldiamines (4) is consistent with the reactivity of this orthoquinone moiety.
The possibility that the increased accumulation of insoluble, crosslinked collagen in fibrotic disease states may be restricted by the specific suppression of lysyl oxidase activity has stimulated the search for selective and potent inhibitors of this enzyme. Thus, this catalyst is subject to mechanism-based inhibition by beta-aminopropionitrile (5) and beta-haloethylamines (6) and is significantly more sensitive to these agents than other mechanistically similar amine oxidases acting on non-peptidyl amine substrates (7).
Certain para-substituted benzylamines (8) and 1,2-alkyldiamines (4) have also been found to be active site-directed inhibitors of lysyl oxidase. Aminoalkylaziridines (figure 1) have been shown to be effective inhibitors of diamine oxidase (9), which, like lysyl oxidase, contains both a copper(II) and a tyrosine-derived quinone cofactor (10,11). N-(4-Aminobutyl)aziridine, a potent inhibitor of cellular polyamine uptake, irreversibly inactivates diamine oxidase (9). These compounds are potentially covalent inactivators in view of their ability to alkylate an enzyme nucleophile at the aziridinyl moiety and to form Schiff base adducts with the carbonyl cofactor at the primary amino group (12-15). Thus, a series of aminoalkylaziridines have been assessed as novel active site directed inhibitors of lysyl oxidase in the present report.
Figure 1. Generic structure of alkylaminoaziridines; "n" = 3 through 7.
3. MATERIALS AND METHODS
3.1 Enzyme purification and assay
Lysyl oxidase was purified to apparent homogeneity from calf aortas as previously described (16). The product migrated as a single band equivalent to a molecular mass of 32,000 Da when analyzed by sodium dodecyl sulfate gel electrophoresis (17). The purified enzyme preparation consists of four, 32 kDa ionic variants exhibiting a high degree of structural similarity, essentially the same substrate specificities and which appear to operate by the same catalytic mechanism (18,19). Aminoalkylaziridines were synthesized and purified as described (20,21).
The specific activity of lysyl oxidase was determined by a tritium release assay against 125,000 cpm of a chick aortic elastin substrate labeled in organ culture with L-(4, 5- 3H)lysine (22). The specific activities of purified enzyme preparations ranged from 400,000 to 800,000 cpm (3H)H2O released per mg-1 protein per 2 h of assay. All activities were corrected for background rates of tritium release of enzyme-free controls, and all activities were fully inhibited by 50 mM b-aminopropionitrile. Enzyme activities were also determined using a peroxidase-coupled fluorometric assay to obtain initial rates of lysyl oxidase-catalyzed H2O2 formation using 2.5 mM 1,5-diaminopentane, or other alkylamines, as specified, in assay mixtures containing 0.25 mg of homovanillic acid, 40 mg of horseradish peroxidase, and 1.2 M urea (23). Assays were initiated by the addition of lysyl oxidase to otherwise complete assay mixtures pre-equilibrated at the optimal assay temperature of 55oC.
4. RESULTS
4.1 Substrate potential of aminoalkylaziridines
The substrate or inhibitory potential of a series of aminoalkylaziridines of varying chain length (figure 2) was assessed in the peroxidase-coupled fluorescence assay. Aziridinyl compounds of different alkyl carbon chain lengths (Figure 1) were each tested at 3 mM in the assay mixtures. Among these, as shown in Figure 2, N-(5-aminopentyl)aziridine (n = 5; figure 1) was the poorest substrate of lysyl oxidase. The similar, marked increase in substrate potential as the chain length was shortened or increased by 1 methylene group (n = 4 or 6, respectively) indicated a uniquely unfavorable substrate effect at n = 5. This variation in the substrate potential of aminoalkylaziridines differed markedly from that obtained with a series of diamine substrates of lysyl oxidase, in that 1,5-diaminopentane and 1,6-diaminohexane (with 5 or 6 methylene groups, respectively, between the two primary amino groups) proved to be most rapidly oxidized at the concentrations tested in the assay system (5 mM). The concentration of diamines (5 mM) used in these assays yielded rates of oxidation which approximated the Vmax for these compounds (6).
Figure 2. Chain length dependency of the substrate potential of diamines and aziridines.
The possibility that N-(5-aminopentyl)aziridine (APAZ) was not only a poor substrate but also an inhibitor of lysyl oxidase was assessed. Indeed, this compound inhibited the activity of the enzyme, using either 1,5-diaminopentane or n-hexylamine as substrate in the peroxidase-coupled fluorescence assay, yielding an IC50 value of 0.5 mM with either substrate. Aminoalkylaziridines with chain lengths shorter or longer than five carbons did not inhibit the enzyme (not shown). Free aziridine did not inhibit the enzyme, indicating both that the inhibition required the primary amino and aziridine moieties and that inhibition was not due to traces of free aziridine possibly present in the preparations of the aminoalkylazirdine.
4.2 Competitive nature of inhibition
A Lineweaver-Burk plot (figure 3) of the data from an initial rate analysis of the mode of inhibition of 1,5-diaminopentane oxidation by APAZ resulted in a series of lines intersecting at the 1/v axis, indicating that the inhibitor competes with the amine substrate for interaction at the active site (figure 3). The KI value calculated from the slopes of these plots was found to be 0.22 mM.
Figure 3. Lineweaver-Burk plot: inhibition of lysyl oxidase by APAZ.
4.3 Irreversibility of inhibition
Lysyl oxidase was preincubated at 37oC in the presence or absence of increasing concentrations of APAZ followed by dilution of aliquots of these mixtures into the peroxidase-coupled assays for 1,5-diaminopentane oxidation, thus reducing the concentration of the aziridinyl compound to non-inhibitory levels in the lysyl oxidase assays. The rates of loss of enzyme activity increased with increasing inhibitor concentrations with inactivation following apparent first order kinetics. As shown (figure 4), the presence of the substrate, 2.5 mM 1,5-diaminopentane, in the preincubation mixture together with the inhibitor partially protected against the loss of enzyme activity caused by the aminoalkylaziridine. The apparent first order rate constant for inactivation (kinact) determined from the intercept of a secondary reciprocal plot (24) of the data of figure 5 was 0.1 min-1. The linearity of this plot is characteristic of an inhibitor exhibiting saturation kinetics. The intercept of the plot at the 1/(I) axis, equal to -1/KI, yielded a KI value of 0.25 mM, closely similar to the value of 0.22 mM determined from the steady-state kinetic experiments described.
Figure 4. Inactivation of lysyl oxidase by APAZ: semi-log plot. Lysyl oxidase was preincubated for the indicated times in the presence of: 0 mM APAZ (control), l; 0.2 mM APAZ, n; 0.5 mM APAZ + 2.5 mM 1,5-diaminopentane (DAP), D; 0.5 mM APAZ, D; 2 mM APAZ, o. Remaining enzyme activity was assayed after dilution (1:50) of aliquots into the peroxidase-coupled assay mixture.
Figure 5. Kitz and Wilson plot. kinact values were calculated from the t1/2 values at each concentration of APAZ (kinact = 0.693/t1/2; see Figure 4).
Consistent with the pseudo first order rate of loss of enzyme activity (figure 4), the inhibition of lysyl oxidase by APAZ was also determined to be irreversible by dialysis. Thus, the enzyme which had been preincubated with 5 mM of the inhibitor at 37oC for 1.5 hours and then dialyzed thoroughly to remove the compound was catalytically inactive, whereas enzyme treated in parallel in the absence of APAZ retained approximately 90% of its original activity.
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 pKa (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 KI and IC50 values 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.
6. ACKNOWLEDGEMENTS
This research was supported by National Institutes of Health grants R37 AR 18880 and HL 13262.
7. REFERENCES
1. H. M. Kagan: Characterization and regulation of lysyl oxidase. In: Biology of the extracellular matrix, Vol l: Regulation of matrix accumulation. Ed: Mecham R P, Academic Press, Orlando (1986)
2. R. C. Siegel: Lysyl oxidase. Int Rev Connect Tiss Res 8, 73-118 (1979)
3. S. X. Wang, M. Mure, K. F. Medzihradszky, A. L. Burlingame, D. Brown, D. M. Dooley, A. J. Smith, H. M. Kagan & J. P. Klinman: A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains. Science 273, 1078-84 (1996)
4. S. N. Gacheru, P. C. Trackman, S. D. Calaman, F. T. Greenaway & H. M. Kagan: Vicinal diamines as PQQ-directed, irreversible inhibitors of lysyl oxidase. J Biol Chem 264, 12963-12969 (1989)
5. S. S. Tang, P. C. Trackman & H. M. Kagan: Reaction of aortic lysyl oxidase with ß-aminopropionitrile. J Biol Chem 258, 4331-4338 (1983)
6. S. S. Tang, D. E. Simpson & H. M. Kagan: ß-Substituted ethylamine derivatives as suicide inhibitors of lysyl oxidase. J Biol Chem 259, 975-979 (1984)
7. S. S. Tang, C. O. Chichester & H. M. Kagan: Comparative sensitivities of purified preparations of lysyl oxidase and other amine oxidases to active site-directed enzyme inhibitors. Conn Tissue Res 19, 93-103 (1989)
8. P. R. Williamson & H. M. Kagan: Electronegativity of aromatic amines as a basis for the development of ground state inhibitors of lysyl oxidase. J Biol Chem 262, 14520-14524 (1987)
9. J. W. Conner, Z. M. Yuan & P. S. Callery: Active-site directed irreversible inhibition of diamine oxidase by a homologous series of aziridinylalkylamines. Biochem Pharmacol 44, 1229-1232 (1992)
10. V. Steinebach, B. W. Groen, S. S. Wijmenga, W. M. A. Niessen, J. A. Jongejan & Duine, J. A.: Identification of topaquinone, as illustrated for pig kidney diamine oxidase and Escherichia coli amine oxidase. Anal Biochem 230, 159-166 (1995)
11. S. M. Janes, M. M. Palcic, C. H. Scaman, A. J. Smith, D. E. Brown, D. M. Dooley, M. Mure & J. P. Klinman: Identification of topaquinone and its consensus sequence in copper amine oxidases. Biochemistry 31, 12147-12154 (1992)
12. J. E. Earley, C. E. O’Rourke, L. B. Clapp, J. O. Edwards &B. C. Lawes: Reaction of ethyleneimines. IX. The mechanisms of ring openings of ethyenimines in acidic aqueous solutions. J Am Chem Soc 80, 3458-3462 (1958)
13. Y. Yamada, T. Imoto & S. Noshita: Modification of catalytic groups in lysozyme. Biochemistry 21, 2187-2192 (1982)
14. C. Weise, H.-J. Kreienkamp, R. Raba, A. Pedak, A. Aaviksaar & F. Hucho: Anionic sites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenylaziridinium. EMBO J 9, 3885-3888 (1990)
15. A. Sepp & J. Järv: Alkylation of acetylcholinesterase anionic center with aziridinium ion accelerates the enzyme acylation step. Biochim Biophys Acta 1077, 407-412 (1991)
16. H. M. Kagan & P. Cai: Isolation of active site peptides of lysyl oxidase. Meth Enzymol 258, 122-132 (1995)
17. U. K. Laemmli: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970)
18. K. A. Sullivan & H. M. Kagan: Evidence for structural similarities in the multiple forms of aortic and cartilage lysyl oxidase and a catalytically quiescent aortic protein. J Biol Chem 257, 13520-13526 (1982)
19. H. M. Kagan, K. A. Sullivan, T. A. Olsson & A. L. Cronlund: Purification and properties of four species of lysyl oxidase from bovine aorta. Biochem J 177, 203-214 (1979)
20. Y. Li, A. D. MacKerell, M. J. Egorin, M. F. Ballesteros, D. M. Rosen, Y.-Y. Wu, D. A. Blamble & P. S. Callery, P: Comparative molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells. Canc. Res. 57, 234-239 (1997)
21. Y.-Y. Wu: Synthesis and evaluation of aziridine-containing compounds as inhibitors of porcine kidney diamine oxidase (Thesis). Baltimore, MD: University of Maryland (1994).
22. H. M. Kagan & K. A. Sullivan: Lysyl oxidase: preparation and role in elastin biosynthesis. Meth Enzymol 82, 637-649 (1982)
23. P. C. Trackman, C. G. Zoski & H. M. Kagan: Development of a peroxidase-coupled fluorometric assay for lysyl oxidase. Anal Biochem 113, 336-342 (1981)
24. R. Kitz & I. B. Wilson: Esters of methanesulfonic acid as irreversible inhibitors of acetylcholine esterase. J Biol Chem 237, 3245-3249 (1962)
25. H. M. Kagan. & P. C. Trackman: Lysyl oxidase. In: Principles and applications of quinoproteins. Ed: Davidson V, Marcel Dekker, NY (1993)
26. H. M. Kagan, M. A. Williams, P. R. Williamson & J. M. Anderson: Influence of sequence and charge on the specificity of lysyl oxidase toward protein and synthetic peptide substrates. J. Biol. Chem. 259, 11203-11207 (1984)