Analysis of the Binding of Hydroxamic Acid and Carboxylic Acid Inhibitors to the Stromelysin-1 (Matrix Metalloproteinase-3) Catalytic Domain by Isothermal Titration Calorimetry

Abstract

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.