PDBsum entry 1i9h

Unknown function

PDB id

1i9h

Contents

			Protein chains

					121 a.a. *

			Ligands

					BNI ×2

			Waters ×134

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Chicken avidin exhibits pseudo-Catalytic properties. Biochemical, Structural, And electrostatic consequences.

Authors

T.Huberman, Y.Eisenberg-Domovich, G.Gitlin, T.Kulik, E.A.Bayer, M.Wilchek, O.Livnah.

Ref.

J Biol Chem, 2001, 276, 32031-32039. [DOI no: 10.1074/jbc.M102018200]

PubMed id

11395489

Abstract

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.



	Figure 7. Fig. 7. Schematic representation of hydrogen-bonding networks of biotin and BNA with streptavidin and avidin as analyzed from their three-dimensional structures. A, in the streptavidin-biotin complex, the biotin carboxylate oxygen forms two H-bond interactions. In contrast, in the avidin-biotin complex (B) the biotin carboxylate oxygens form five H-bond interactions, three of which are contributed from residues belonging to the L3,4 loop. In the streptavidin-BNA complex (C), the H-bond network is identical to that of the streptavidin-biotin complex (A). In the avidin-BNA complex (D), however, the L3,4 adopts an open, partially disordered conformation as opposed to that of the avidin-biotin complex. The open conformation of L3,4 results in the loss of three H-bonds with the BNA amide group. In addition, Thr-35 is displaced, resulting in the loss of another H-bond between the O and one of the uredio nitrogens of the ligand. Most significantly, the conformation of the p-nitroanilide moiety in the avidin-BNA complex results in a decisive interaction between its nitro group and the N of Lys-111 from an adjacent monomer.		Figure 9. Fig. 9. Mechanism of BNP hydrolysis by avidin. The conformation of the BNP ligand facilitates both interaction with Lys-111 from an adjacent monomer and accessibility to attack by an OH ion. Interaction of the nitro group of the ligand with Lys-111 increases the electron-withdrawing properties of the p-nitrophenyl moiety, thereby promoting hydrolysis at relatively low pH.

PROCHECK

	Headers