PDBsum entry 1knh

Hydrolase

PDB id: 1knh

Contents

Protein chain

198 a.a.

Ligands

VAL-ALA-ALA-LEU-TYR-LEU-VAL-CYS-GLY-ALA

Metals

_CA

_ZN

Superseded by:

1m1v

Theoretical model

PDB id:

1knh

Name:

Hydrolase

Title:

Molecular dynamics of mmp-3, adam-9 and adam-10: new implications on familiarity, structure, function and substrate affinity

Structure:

Meltrin gamma. Chain: a. Fragment: catalytic domain. Synonym: adam-9. A disintegrin and metalloproteinase domain 9. Metalloprotease/disintegrin/cysteine-rich protein. Empirical substrate. Chain: b. Engineered: yes

Source:

Homo sapiens. Human. Synthetic: yes

Authors:

S.Manzetti,D.R.Mcculloch,A.C.Herington

Key ref:

F.H.Vaillancourt et al. (1998). Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol. J Biol Chem, 273, 34887-34895. PubMed id: 9857017 DOI: 10.1074/jbc.273.52.34887

Date:

18-Dec-01 Release date: 04-Jan-02

Protein chain

No UniProt id for this chain

Struc: 198 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.24.17 - stromelysin 1.
• Reaction: Preferential cleavage where P1', P2' and P3' are hydrophobic residues.
• Cofactor: Ca(2+); Zn(2+)

DOI no: 10.1074/jbc.273.52.34887

J Biol Chem 273:34887-34895 (1998)

PubMed id: 9857017

Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol.

F.H.Vaillancourt, S.Han, P.D.Fortin, J.T.Bolin, L.D.Eltis.

ABSTRACT

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1, 2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for DHB were 22 +/- 2 and 8 +/- 1 microM, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.

Selected figure(s)

Figure 3.
Fig. 3. Relationship of the two t-butanol-binding sites to groups at the active site. A, in the free enzyme, t-butanol occupies the distal portion of the substrate-binding site. B, in the enzyme-substrate complexes, t-butanol occupies an auxiliary site adjacent to the distal portion of the substrate-binding site, further removed from the iron.

Figure 4.
Fig. 4. Electron density maps and models illustrating the structure of the DHBD:3-methylcatechol-bound and substrate-free forms of DHBD. Each part is a (divergent) stereo drawing prepared with the program MolView (42). The identical observed structure factors were used in all maps, which demonstrates the presence of both forms of the enzyme in the same crystal. All maps are at 1.9-Å resolution and are contoured at two standard deviations above the mean of the map. In the models, the carbon atoms are more darkly shaded than the nitrogen and oxygen atoms. A, F[o] F[c] electron density representing the iron, 3-methylcatechol, and two water ligands. The F[c] 's and phases are from the structure of the substrate-free enzyme (3). The model is the initial fit to this density. B, residual F[o] F[c] electron density following refinement of a model that included the iron, 3-methylcatechol, two water ligands, and a t-butanol bound in the auxiliary site, as shown. The F[c] 's and phases are from this model. The density features arise from the fraction of the crystal that is in the substrate free-form, as demonstrated by C, which shows the refined model of this form (3) in conjunction with the same density. Note that the t-butanol binds in the substrate-binding site in this form of the enzyme.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (1998, 273, 34887-34895) copyright 1998.

Figures were selected by an automated process.