Interactions of Human Matrix Metalloproteinase 7 (Matrilysin) with the Inhibitors Thiorphan and R-94138

Abstract

The effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysin-catalyzed hydrolysis of (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) [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (K(i)) of thiorphan and R-94138 for matrilysin at pH 7.5, 25 degrees C were determined to be 11.2 and 7.65 microM, respectively. From the temperature dependence of the K(i) values at pH 7.5, the standard enthalpy change (Delta H degrees ') values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2 +/- 0.9) and (1.65 +/- 1.07) kJ x mol(-1), respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the K(i) value suggests that at least two ionizing groups with pK(a) values of 4.5 and 9.1--9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pK(a) values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.

Neutral endopeptidase inhibition in diabetic wound repair

“…However, thiorphan has recently been shown to bind other metalloproteases including matrix metalloproteinase 7 (matrilysin). 32 Matrilysin expression is up-regulated in corneal basal epithelial cells after wounding and has been speculated to be involved in epithelial migration and proliferation. 33 Therefore, an interaction between matrilysin and thiorphan may have contributed to our observed improvement in wound closure kinetics.…”

Anomalous pH-dependence of the activity of human matrilysin (matrix metalloproteinase-7) as revealed by nitration and amination of its tyrosine residues

“…We have reported the high stability and denaturation properties of matrilysin [12,13]. The interaction of matrilysin with synthetic inhibitors has been studied, and the significance of hydrophobic interactions has been suggested in the recognition of the inhibitors at the active site of matrilysin [14]. Green tea catechins, especially those with a galloyl group, inhibit matrilysin in non-competitive manner with a K i of 0.47-1.65 µM [15].…”

“…The pH-dependence of matrilysin activity exhibits an extraordinarily broad bell-shaped curve, indicating the involvement of at least two ionizable groups in its catalytic mechanism [14,16]. (the numbering of the amino acid residues of pro-matrilysin is applied in this paper to activated matrilysin, beginning at number 78 for the N-terminal tyrosine residue [17]) or a water molecule bound to the active zinc (Lewis acid) is considered to be the ionizable group of the acidic side with pK e1 [18][19][20].…”

“…(In this paper, the pK a of the ionizable group on matrilysin is designated pK e , and that on the enzyme-substrate complex is pK e ; the suffixes 1 and 2 indicate that the respective pK a s are on the acidic and alkaline sides respectively.) On the other hand, Tyr-219, which is conserved in all MMPs [21], is thought to control activity as the group responsible for pK e2 [14,16], although no decisive evidence for this has been reported. The objectives of the present study were to elucidate the functional groups and to provide insights into the reaction mechanism of matrilysin.…”

Effects of heparin and cholesterol sulfate on the activity and stability of human matrix metalloproteinase 7

“…4) The development of MMP-7 inhibitors is thought to be of potential therapeutic benefit. We have reported that alcohols, 12) synthetic MMP inhibitors thiorphan and R-94138, 13) and lignans 14) inhibit MMP-7 activity in a competitive manner, while green tea catechins 15,16) and a fluorescent probe, 8-alininonaphthalene 1-sulfonate (ANS), 17) inhibit it in a non-competitive manner. MMP-7 exhibits a broad bell-shaped pH profile of MMP-7 activity with acidic and alkaline pK a (pK e1 and pK e2 ) values of about 4 and 10.…”