title: Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7).
abstract: 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. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. pK(a) of side chains of L-Asp, L-Glu, L-His, L-Cys, L-Tyr, L-Lys, and L-Arg at 25-45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ∆H°, of deprotonation were calculated. pK(e1) and pK(e2) of MMP-7 at 15-45°C were determined 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-NH(2), from which ∆H(o) for pK(e1) and pK(e2) was calculated. The ∆H(o) for pK(e1) (-20.6±6.1kJmol(-1)) was similar to that for L-Glu (-23.6±5.8kJmol(-1)), and the ∆H(o) for pK(e2) (89.9±4.0kJmol(-1)) was similar to those for L-Arg (87.6±5.5kJmol(-1)) and L-Lys (70.4±4.4kJmol(-1)). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for pK(e1). On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for pK(e2).
authors.affiliation: Kyoto University, Kyoto, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takeharu
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D006801:Humans; D007477:Ions / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D007477:Ions; D050505:Mutant Proteins; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7