Abstract
The crystal structure of the binary complex of nonactivated ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and a transition state analogue, 2-carboxy-D-arabinitol 1,5-bisphosphate has been determined to 2.6 A resolution with x-ray crystallographic methods. The transition state analogue binds in a rather extended conformation at the active site. The orientation of the transition state analogue within the active site could be determined from the electron density maps. The P1 phosphate group of the analogue binds at a site built up of residues from loops 5 and 6 of the alpha/beta-barrel. The phosphate group interacts with the side chains of the conserved residues Arg-288, His-321, and Ser-368 and with main chain nitrogens from residues Thr-322 and Gly-323. The second phosphate group of the transition state analogue binds at the opposite side of the barrel close to loops 1 and 8. Significant differences for the positions and interactions of the P2 phosphate group with the enzyme are found in the two subunits of the dimer. The different mode of binding for this phosphate group in the two subunits is interpreted as a consequence of different conformations of the polypeptide chain observed in loops 6 and 8. The P2 phosphate group interacts with the sidechains of Lys-166 and Lys-329. Loop 6, which is disordered in the nonactivated, nonliganded enzyme is considerably more ordered in one of the subunits, probably due to the interaction of the side chain of Lys-329 with the P2 phosphate group. Almost all oxygen atoms are hydrogen bonded to groups on the enzyme. The carboxyl group forms hydrogen bonds to the side chain of the conserved Asn-111. The binding of the transition state analogue to the nonactivated enzyme is different from the binding of the analogue to activated spinach ribulose-bisphosphate carboxylase.
Highlights
The crystal structureof the binarycomplex of non- conserved lysine side chain (Lys-191 in the Rhodospirillum activated ribulose-1,5-bisphosphatecarboxylase/oxy- rubrum enzyme) with a carbon dioxidemolecule with the genase from Rhodospirillum rubrum and a transition formation of a carbamate
The first partial reaction, the enolization of ribulose biszyme are found in the two subunitosf the dimer
The phosphate can be followed by the “wash out” of radioactivity different mode of binding for this phosphate group in from [3-3H]ribulosebisphosphate (Sue and Knowles, 1982)
Summary
The first andsecond partial reactions arecatalyzed by the activated wild-type enzyme, whereas thethird reaction is chain of Lys-329 with thPe 2 phosphate group.Almost catalyzed by the nonactivated carboxylase. This all oxygen atomasre hydrogen bonded to grouposn the is so far the only activity known to be associated with the enzyme. Activation consists of the reaction of the eamino group of a lose-bisphosphate carboxylase from R. rubrum (Schneider et al, 1986) and tobacco (Chapman et al 1987; Chapman et al, 1988).The crystal structureof the activated quaternarycomplex ofspinach ribulose bisphosphate carboxylase with bound transitionstate analogue CA-P2has beensolved recently (Anderson et al, 1989) From these crystallographic studies, a detailed picture of the active site of the enzyme has begun to emerge. ’ The abbreviations used are: KCA-P,, 2-carboxy-3-keto-~-arabinitol-1,5-bisphosphate; ribulose-Pz carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase; CA-P2,2-carboxy-~-arabinitol-1,5-bisphosphate; 3-P-glycerate, 3-~-phospho-glycerate;MPD, 2-methyl2,4-pentanediol; F,; observed structure factor amplitude; F,, calculated structure factor
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