Analysis of the Binding of Hydroxamic Acid and Carboxylic Acid Inhibitors to the Stromelysin-1 (Matrix Metalloproteinase-3) Catalytic Domain by Isothermal Titration Calorimetry
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- 作者：Matthew H. Parker ; Elizabeth A. Lunney ; Daniel F. Ortwine ; Alexander G. Pavlovsky ; Christine Humblet ; and Christie G. Brouillette
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：October 12, 1999
- 年：1999
- 卷：38
- 期：41
- 页码：13592 - 13601
- 全文大小：109K
- 年卷期：v.38,no.41(October 12, 1999)
- ISSN：1520-4995
文摘
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 anattractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze thebinding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses ahydroxamic acid group (pKa 
8.7) to bind the active site zinc; the others (PD180557 and PD166793)use a carboxylic acid group (pKa 4.7). Binding affinity increased dramatically as the pH was decreasedover the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed thatapproximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamicand carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in theprotonated 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 histidine224. 
G, H, S, and Cp were determined for the three inhibitors at pH 6.7, and Cp was used toobtain 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' substituentsof 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 discoveringbinding interactions that might go undetected when relying on binding affinities alone.
8.7) to bind the active site zinc; the others (PD180557 and PD166793)use a carboxylic acid group (pKa 4.7). Binding affinity increased dramatically as the pH was decreasedover the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed thatapproximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamicand carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in theprotonated 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 histidine224. G, H, S, and Cp were determined for the three inhibitors at pH 6.7, and Cp was used toobtain 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' substituentsof 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 discoveringbinding interactions that might go undetected when relying on binding affinities alone.