Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK(e) (pK(e1) and pK(e2)) values of about 4 and 10. Its active-site tyrosyl residue, Tyr219, is conserved in all other MMPs, and thus has been thought for the ionizable group responsible for pK(e2). In this study, we examined the mutational effects of Tyr219 on enzyme activity. Five Tyr219 variants, Y219F (Tyr219 is replaced with Phe), Y219D, Y219A, Y219C and Y219S, were constructed by site-directed mutagenesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2, all five variants retained the activity, indicating that Tyr219 is not the ionizable group responsible for pK(e2). Unexpectedly, all five variants exhibited narrower pH-dependence than the wild-type MMP-7, with the pK(e1) and pK(e2) values in the range of 5.2-5.4 and 8.6-9.4, respectively. Such pH-dependence shifts were not observed in other active-site tyrosyl-residue variants, Y193F and Y216F. These results suggest that Tyr219 is not critical for catalytic activity, but is involved in the broad pH-dependence of the activity.

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).

Matrilysin activity exhibits a broad bell-shaped pH-dependence profile, with pK(a) values of 4.0 and 9.8. A maximum of five out of eight tyrosine residues in matrilysin were nitrated with tetranitromethane. On nitration of between one and five tyrosines, pK(a) at the alkaline side (pK(e2)) was shifted from 9.8 to 10.3-10.6, while that at the acidic side (pK(e1)) was not altered. The pK(e2) that was shifted by nitration to 10.3-10.6 was restored to 9.4-9.7 by subsequent amination, suggesting that the shift in pK(e2) is induced by a negative charge introduced on the most reactive tyrosine, Tyr-150. The Michaelis constant (K(m)) observed at pH 10 was decreased by nitration as a result of the increase in pK(e2), suggesting that the residue with pK(e2) may play a role in the recognition of substrate. When four or five tyrosines were nitrated, the activity at pH <7 decreased significantly, while that at pH 7-10 was unchanged, and thus the pH-dependence was not bell-shaped, but anomalous, with a third pK(a) (pK(e3)) of 6.2-6.4 in addition to pK(e1) and pK(e2). This suggests the possibility that a newly introduced nitrotyrosine residue has a strong influence on the activity as an ionizable group.

Inhibitory effects of nine dibenzylbutyrolactone lignans on a human matrix metalloproteinase, matrilysin, were examined. All of the lignans examined inhibited matrilysin with the IC(50) values ranging from 50 to >280 microM. Matairesinol, which has the basic structure of the other lignans, showed the weakest inhibition. Lignans with methylenedioxy ring(s) or a hydroxyl group at the C5-position inhibited matrilysin more strongly than matairesinol. 5-Hydroxypluviatolide, which has both a methylenedioxy ring and a hydroxyl group at the C5-position, was the most potent inhibitor (IC(50) = 50 microM), suggesting that the introduction of these two elements might enhance synergistically the inhibitory activity of lignans. 5-Hydroxypluviatolide inhibited matrilysin in a competitive manner, and its inhibitory effect was greatly suppressed by the presence of another competitive inhibitor, dimethyl sulfoxide. The precursors of matairesinol, coniferyl alcohol and secoisolariciresinol, had no inhibitory activity, indicating that the dibenzylbutyrolactone structure is essential for the inhibition. It has been shown that lignans have the potential to inhibit matrilysin, and the knowledge of their structure-activity relationship might be beneficial to developing selective inhibitors for matrix metalloproteinases.

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with K-i of 6.1-19.4% (v/v) or 0.66-4.80M. from the relationship between the structures of alcohols and their K-i values, alcohols are considered to bind the hydrophobic S1' subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.

Salt-activation of thermolysin was examined using a positively charged fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 [MOCAc-PLGL(Dpa)AR]. Thermolysin activity increased in a biphasic exponential fashion and was 40 times higher in the presence of 4 M NaCl than in its absence. The degree of activation at x M NaCl was expressed as 4.7x when [NaCl]o<0.5 M and 2.3x when [NaCl]o>0.5 M respectively.

Thermal unfolding of P. cepacia lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme. The temperature of unfolding was higher at higher concentrations of Ca2+. From the Ca2+ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one. These results confirmed the validity of the unfolding mechanism proposed previously: NCa2+ < = => D + Ca2+, where N and D represent the native and denatured states, respectively, of the enzyme.

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH(2) and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2). The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K(i)) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol >> tert-butyl alcohol >> methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.