E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

Linda Troeberg, Mitsuo Tanaka, Robin Wait, Yeunian E Shi, Keith Brew, Hideaki Nagase

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.

Original languageEnglish
Pages (from-to)15025-1535
Number of pages11
JournalBiochemistry
Volume41
Issue number50
Early online date16 Nov 2002
DOIs
Publication statusPublished - 17 Dec 2002

Keywords

  • Amino Acid Sequence
  • Animals
  • Binding, Competitive
  • CHO Cells
  • Cricetinae
  • Escherichia coli/genetics
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Matrix Metalloproteinase 2/metabolism
  • Matrix Metalloproteinase Inhibitors
  • Molecular Sequence Data
  • Protein Folding
  • Recombinant Proteins/biosynthesis
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tissue Inhibitor of Metalloproteinase-1/metabolism
  • Tissue Inhibitor of Metalloproteinase-2/metabolism
  • Tissue Inhibitor of Metalloproteinases/biosynthesis
  • alpha-Macroglobulins/chemistry

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