Ribulose diphosphate oxygenase. V. Presence in ribulose diphosphate carboxylase from Rhodospirillum rubrum

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H₂ and CO₂. There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg⁺⁺ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.     

Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.     

Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.     

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.     

Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase

Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).     

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.