The role of the allosteric B site in the fumarase reaction

Abstract

The role of a malate binding site in a concavity external to the more deeply situated active site has been a major mystery of the fumarase reaction. The malate, within 12 A of the active site, was bound by hydrogen bonds to two main-chain amides and to two basic residues, H129 and R126. Mutation of the His of this so-called B site of Escherichia coli fumarase had little effect on the overall initial rate kinetics of the enzyme, which has obscured an understanding of the critical role of the site. Contrary to the WT enzyme, which is rate-limited in the recycling of free enzyme isoforms that follows product release, the enzyme with both basic residues modified is rate-limited in the product release step itself. A loss of complexity in the mutated, but still functional, step is indicated by a greatly reduced sensitivity of its rate to changes in temperature. Unlike the inhibition by glycerol shown with normal enzyme and attributed to a viscogenic effect on the recycling rate, the product-release step of the B-site mutants is accelerated by glycerol, suggestive of a structural effect on the 12-A space between the A and B sites. It is proposed that the "extra" malate represents a stage in the transfer of substrate and product between the solvent and the "buried" active site of the enzyme.

Fig. 1.

A and B sites within FumC. The concavity opening to the A and B sites is illustrated in ball-and-stick fashion. The A site, identified by the location of both the citrate and water 26 (W-26) molecules, lies at the back of the opening ≈20 Å from the enzyme surface. The B site is located ≈12 Å from the A site and identified by the position of the bound M. Dashed lines in the drawing denote hydrogen bonds. Each residue has been labeled to identify the subunit from which it is derived. Atoms are colored as follows: carbon (gray), nitrogen (dark gray), and oxygen (black). The illustration was made with the chimera package from the Computer Graphics Laboratory, University of California, San Francisco [supported by National Institutes of Health Grant P41 RR-01081 (www.cgl.ucsf.edu/chimera)].

Scheme 1.

Fig. 2.

ΔT of F → M with WT and DM. (A) K127D (like WT) is rate-limited in recycling (in 20 mM Tris acetate, pH 7.5), ^Δ20°V_max = 1.9 and ^Δ20°Slope = 1.0. The units for v are ΔA/min. (B) DM is about the same at 20°C and 35°C.

Fig. 3.

ΔT of WT and DM in M → F. (A) WT, 20 mM Tris acetate, pH 7.56, ^Δ15°V_max = 1.75 and ^Δ15°Slope = 1.0. (B) DM, 10 mM Tris acetate + 50 mM sodium acetate, pH 7.8, ^Δ20°V_max = 1.8 and ^Δ20°Slope = 1.6.

Fig. 4.

Effect of glycerol on WT and DM rates. (A) WT in 10 mM Tris chloride, pH 7.2, and F from 0.025 to 0.15 mM. (B) DM in 10 mM 2-amino-2-methyl-1-propanol, pH 9.0, and F from 0.2 to 4 mM. (C) DM in 20 mM Tris acetate + 50 mM sodium acetate, pH 7.5, and M from 5 to 40 mM.