Heather J. Kane

Substrate isomerization inhibits ribulosebisphospate carboxylase‐oxygenase during catalysis

The inhibition of purified spinach ribulosebisphosphate carboxylase‐oxygenase which occurs progressively during catalysis in vitro is caused by accumulation of at least two tight‐binding inhibitors at the catalytic site. Reduction of these inhibitors with NaB3H4, followed by dephosphorylation, produced a mixture of xylitol and arabinitol, thus identifying one of them as D‐xylulose 1,5‐bisphosphate. It was formed during carboxylation, presumably by a stereochemically incorrect reprotonation of the 2,3‐enediolate intermediate bound at the catalytic site. Under the conditions used, this epimerization occurred approximately once for every 400 carboxylation turnovers. Another inhibitor may be 3‐keto‐D‐arabinitol 1,5‐bisphosphate which would also be formed by misprotonation of the enediolate intermediate, but at C‐2 rather than at C‐3.

Measurement of (carbon) kinetic isotope effect by Rayleigh fractionation using membrane inlet mass spectrometry for CO2-consuming reactions

Methods for determining carbon isotope discrimination, Δ, or kinetic isotope effects, α, for CO2-consuming enzymes have traditionally been cumbersome and time-consuming, requiring careful isolation of substrates and products and conversion of these to CO2 for measurement of isotope ratio by mass spectrometry (MS). An equation originally derived by Rayleigh in 1896 has been used more recently to good effect as it only requires measurement of substrate concentrations and isotope ratios. For carboxylation reactions such as those catalysed by d-ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO, EC 4.1.1.39) and PEP carboxylase (PEPC, EC 4.1.1.31), this has still required sampling of reactions at various states of completion and conversion of all inorganic carbon to CO2, as well as determining the amount of substrate consumed. We introduce a new method of membrane inlet MS which can be used to continuously monitor individual CO2 isotope concentrations, rather than isotope ratio. This enables the use of a simplified, new formula for calculating kinetic isotope effects, based on the assumptions underlying the original Rayleigh fractionation equation and given by.

Metabolic studies on the new fasciolicidal drug, closantel

Studies of the metabolic disturbances caused in Fasciola hepatica by closantel have been carried out in vitro and in fluke recovered from treated sheep. Fluke exposed to the anthelmintic under both conditions exhibit increased carbohydrate mobilisation, increased end-product formation, especially of succinate, diminished ATP synthesis, increased oxaloacetate/malate ratios, and increased internal concentrations of pyruvate. No specific enzyme inhibition was detected. These results are consistent with the view that closantel acts as an uncoupler of oxidative phosphorylation by increasing mitochondrial permeability.

Side reactions catalyzed by ribulose-bisphosphate carboxylase in the presence and absence of small subunits.

The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the kcat of the holoenzyme) (Andrews, T. J (1988) J. Biol. Chem. 263, 12213-12219). During steady-state catalysis at substrate saturation, this residual activity diverted approximately 10% of the reaction flux to 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of β elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate. This indicates that the active sites ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits. Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits. The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in a ratio similar to that of the holoenzyme, indicating that stablization of the penultimate three-carbon aci-acid intermediate is not perturbed by lack of small subunits. The underlying instability of the five-carbon enediol intermediate was revealed, even with the holoenzyme, under conditions designed to lead to exhaustion of substrate CO2 (and O2). When carboxylation (and oxygenation) stalled upon exhaustion of gaseous substrate, both spinach and Synechococcus holoenzymes continued slowly to β eliminate inorganic phosphate from and to misprotonate the enediol intermediate. With carboxylation and oxygenation blocked, the products of these side reactions of the enediol intermediate accumulated to readily detectable levels, illustrating the difficulties attendant upon ribulose-P2 carboxylases use of this reactive species as a catalytic intermediate.

High temperature enhances inhibitor production but reduces fallover in tobacco Rubisco

High temperature inhibits photosynthesis by several mechanisms including reduction in Rubisco activity. While the initial reaction velocity of purified, fully carbamylated, inhibitor-free Rubisco increases with temperature in vitro, over time, the reaction velocity slowly declines (fallover) because of the enzymatic and non-enzymatic production of inhibitors from the substrate ribulose-1,5-bisphosphate. We tested whether fallover could contribute to the decline in Rubisco activity observed in leaf extracts at high temperature. Production of d-xylulose-1,5-bisphosphate (XuBP), an inhibitor of Rubisco, was greater at 35 and 45°C than at 25°C but fallover was less severe at 35 and 45°C than at 25°C, both in rate and extent under saturating CO2 and ambient O2. This apparent dichotomy is consistent with the catalytic site of Rubisco loosening at higher temperatures and releasing inhibitors more easily. The loosening of the catalytic site was supported by the observation that RuBP and XuBP were released from their complexes with uncarbamylated, Mg2+-free Rubisco faster at 35 and 45°C than at 25°C. We conclude that, although XuBP production increased relative to catalytic throughput at higher temperatures, this was more than compensated for by its faster release, resulting in less fallover inhibition at higher temperatures.

Carboxylterminal deletion mutants of ribulosebisphosphate carboxylase from Rhodospirillum rubrum

The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphosphate carboxylases, which are composed of large and small subunits. Truncations of the R. rubrum enzyme, which partially or completely deleted the region of homology, demonstrated that the region is not an important determinant of the catalytic efficiency of the enzyme. A further truncation, which replaced the carboxylterminal 19 amino acid residues with a single terminal leucyl residue, yielded a Rubisco whose substrate‐saturated catalytic rate resembled that of the wild‐type enzyme but which had weaker affinities for ribulose‐P2 and CO2.

Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301

Previous studies [G. S. Hudson et al. (1989) J. Biol. Chem. 265, 808-814] showed that the faster turnover rates and lower affinities for CO2 of ribulosebisphosphate carboxylase/oxygenases from C4 plants, compared to C3 and C3/C4 plants, were specified by the chloroplast-encoded large subunits. In pairs of closely related C3 and C4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus. None of these substitutions occurred near the active site and only one, 309Met (C3) to Ile (C4), was common to all three genera. Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli. Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO2. 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL. Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme. However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits. The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked.

3-Mercaptopicolinic acid and energy metabolism in the liver fluke, Fasciola hepatica.

Abstract Mercaptopicolinic acid inhibited 14 CO 2 uptake and phosphoenolpyruvate carboxykinase activity in intact fluke. Studies with enzyme preparations showed that the inhibition was mixed-competitive with phosphoenolpyruvate and non-competitive with GTP. Inhibition was not reversed by Mn 2+ . Pyruvate kinase was not inhibited by mercaptopicolinic acid, although under certain circumstances, mercaptopicolinic acid interfered with the pyruvate kinase assay system. Intact flukes incubated with mercaptopicolinic acid showed depressed adenylate energy charge, increased lactic acid production and reduced flow of carbon from phosphoenolpyruvate to the mitochondrial substrate, malate. Additions of glutamate, alanine or aspartate did not reverse these effects even though, in each case, the amino acid was metabolised and considerably more acid end products were formed than in the absence of mercaptopicolinic acid. The changes in the concentrations of metabolites and end products are consistent with the view that, in flukes whose energy metabolism is impaired by mercaptopicolinic acid, pyruvate enters the mitochondrion and is converted to acetic and propionic acids.

Rubisco: Subunits and Mechanism

D-ribulose-l,5-bisphosphate (ribulose-P2) carboxylase-oxygenase (Rubisco) catalyses the initial reactions of the photosynthetic carbon reduction cycle and its photorespiratory appendage, the glycolate pathway, in all photosynthetic organisms. Except within the Athiorhodaceae, all Rubiscos are complex hexadecameric proteins composed of eight large (L) subunits, which bear the catalytic sites, and eight small (S) subunits, whose function is the subject of considerable current inquiry (1). Recently, this inquiry has been aided by the availability of structural information from crystallographic studies (2,3), by techniques for reversibly separating the large and small subunits of cyanobacterial Rubiscos (1) and by expression of the polypeptide products of cyanobacterial rbcL and rbcS genes in Escherichia coli (4,5). Insights into the interactions between the large and small subunits obtained by these approaches will be reviewed and the characteristics of the isolated subunits reported.