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

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 V_max 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 K_I 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 37^oC 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 (k_inact) 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/K_I, yielded a K_I 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. k_inact values were calculated from the t_1/2 values at each concentration of APAZ (k_inact = 0.693/t_1/2; 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 37^oC 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.