Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

Abstract

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

The three‐dimensional structures of mutants of porphobilinogen deaminase: Toward an understanding of the structural basis of acute intermittent porphyria

“…Residues R149, R150, R167, and R173 [131, 132, 149, and 155 in E. coli] all provide stabilizing interactions with the acetate and propionate side groups of the cofactor in the active site as shown in Figure 2A. Residue R149 [I311 interacts with side chains from ring C1 of the cofactor, and its change by site-directed mutagenesis to H131 or L131 in the E. coli enzyme prevents cofactor assembly and results in an inactive apo-enzyme (Jordan & Woodcock, 1991;Lander et al, 1991). The E. coli apo-enzyme is unstable (Scott et al, 1989) and the human enzyme is probably rapidly degraded, thus explaining the CRIM-status of the 149 [I311 mutants.…”

“…In many ways our analysis is inspired by and follows the approach developed by Max Perutz in analyzing mutants of human hemoglobin. This approach is further aided by the characterization of site-directed mutants of the E. coli enzyme, designed to probe the enzyme mechanism, and some of which have direct counterparts in AIP-associated human mutants (Jordan & Woodcock, 1991;Lander et al, 1991).…”

“…The structure possesses a large number of other ion pairs that may contribute to the considerable heat stability of the enzyme. Enzyme-intermediate complexes, generated as the 4 molecules of substrate are added sequentially to the cofactor primer, are stable to various degrees and may be isolated (Lander et al, 1991;Warren & Jordan, 1988). The escalating negative charge that develops as the pyrrole chain is elongated has focused attention on the role of the highly conserved basic residues in the protein.…”

“…The escalating negative charge that develops as the pyrrole chain is elongated has focused attention on the role of the highly conserved basic residues in the protein. Site-directed mutagenesis of several of these residues results in the accumulation of distinct patterns of intermediates in the polymerization reaction, with some mutations (at R131 and R132) preventing the formation of the co- factor (Lander et al, 1991;Jordan & Woodcock, 1991). There is evidence that relative movement of the domains may be important during the catalytic cycle to accommodate the intermediates (Warren & Jordan, 1988).…”

Structure and Expression of Chloroplast-Localized Porphobilinogen Deaminase from Pea (Pisum sativum L.) Isolated by Redundant Polymerase Chain Reaction

“…A number of conserved residues have been shown by chemical modification studies or site-directed mutagenesis of the Escherichia coli enzyme to be important for catalytic activity. Among these conserved residues is CysZ4', which binds the dipyrromethane cofactor Miller et al, 1988), severa1 Arg's involved in substrate binding (Jordan and Woodcock, 1991;Lander et al, 1991), and putative active site Lys's (Hadener et al, 1990). The recent determination of tbe three-dimensional structure of the E. coli enzyme at 1.9 A resolution (Louie et al, 1992) will greatly facilitate the study of structure-function relationships in this enzyme.…”