Role of inosine 5'-phosphate in activating glucose-bisphosphatase

Glucose-bisphosphate (G1c-1,6-P2) phosphatase has been purified greater than 200-fold from the cytosol of mouse brain. As reported earlier, the enzyme requires inosine monophosphate (IMP) and Mg2+ for activity [Guha, S.K., & Rose, Z. B. (1982) J. Biol. Chem. 257, 6634-6637]. Kinetic parameters and the role of IMP have been further investigated. When Glc-1,6-P2 and IMP are both varied, double-reciprocal plots of the data form a parallel line pattern. With 2 mM Mg2+, the Km obtained for G1c-1,6-P2 is 20 microM and the Ka for IMP is 9 microM. Co2+, Mn2+, and Ni2+ activate less effectively than Mg2+. The apparent Ka for Mg2+ decreases with increasing G1c-1,6-P2, and the observed Km of G1c-1,6-P2 decreases with increasing Mg2+. The extrapolated value of the Ka of Mg2+ at infinite substrate is 86 microM. Mg2+ does not affect the Ka of IMP. The phosphatase activity is optimal at pH 7. The phosphatase is not completely specific since mannose 1,6-bisphosphate is hydrolyzed and guanosine monophosphate activates. However, fructose 1,6-bisphosphate is no more than a poor inhibitor, and adenine nucleotides are neither activators nor inhibitors. The products of the reaction are glucose-1-P and glucose-6-P, in a ratio of 2:3, and Pi. Both glucose-P's are competitive inhibitors with respect to IMP [Ki(glucose-1-P) = 5 microM; Ki(glucose-6-P) = 18 microM]. Neither glucose-P competes with G1c-1,6-P2. The demonstration of an exchange reaction between G1c-1,6-P2 and glucose-6-P is evidence for the phosphorylation of the enzyme by the substrate. The exchange reaction requires Mg2+ and is inhibited by IMP. The observation of the exchange reaction and its elimination by IMP indicates that the low level of phosphoglucomutase activity that remains with the phosphatase throughout purification is an inherent property of the phosphatase. The requirement of glucose-bisphosphatase for the nucleotide IMP is consistent with possible roles for both G1c-1,6-P2 and IMP in the control of the ATP level in the brain.     

Indiscriminate binding by orotidine 5'-phosphate decarboxylase of uridine 5'-phosphate derivatives with bulky anionic c6 substituents

Orotidine 5'-phosphate (OMP) decarboxylase appears to act upon its substrate without the intervention of metals or other cofactors and without the formation of covalent bonds between the enzyme and the substrate. Crystallographic information indicates that substrate binding forces the substrate's scissile carboxylate group into the neighborhood of several charged groups at the active site. It has been proposed that binding might result in electrostatic stress at the substrate's C6 carboxylate group in such a way as to promote decarboxylation by destabilizing the enzyme-substrate complex in its ground state. If that were the case, one would expect uridine 5'-phosphate (UMP) derivatives with bulky anionic substituents at C6 to be bound weakly compared with UMP, which is unsubstituted at C6. Here, we describe the formation of anionic 5,6-dihydro-6-sulfonyl derivatives by spontaneous addition of sulfite to UMP and to OMP. These sulfite addition reactions, which are slowly reversible and are not catalyzed by the enzyme, result in the appearance of one (or, in the case of OMP, two) bulky anionic substituents at the 6-carbon atom of UMP. These inhibitors are bound with affinities that surpass the binding affinity of UMP. We are led to infer that the active site of OMP decarboxylase is remarkably accommodating in the neighborhood of C6. These are not the properties that one would expect of an active site with a rigid structure that imposes sufficient electrostatic stress on the substrate to produce a major advancement along the reaction coordinate.     

Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.     

Characterization of the pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate hydrolase activity in rat liver. Identity with alkaline phosphatase.

The tissue content of pyridoxal 5'-phosphate is controlled principally by the protein binding of this coenzyme and its hydrolysis by a cellular phosphatase. The present study identifies this enzyme and its intracellular location in rat liver. Pyridoxal-P is not hydrolyzed by the acid phosphatase of intact lysosomes. At pH 7.4 and 9.0, the subcellular distribution of pyridoxal-P phosphatase activity is similar to the for p-nitrophenyl-P, and the major portion of both activities is found in the plasma membrane fraction. The ratio of specific activities for pyridoxal-P and p-nitrophenyl-P hydrolysis remains relatively constant during the isolation of plasma membranes. These activities also behave concordantly with respect to pH rate profile, pH-Km profile, and response to chelating agents, Zn2+, Mg2+, and inhibitors. Kinetic studies indicate that pyridoxal-P binds to same enzyme sites as beta-glycerophosphate and phosphorylcholine. The data strongly favor alkaline phosphatase as the enzyme which functions in the control of pyridoxal-P and pyridoxamine-P metabolism in rat liver. Alkaline phosphatase was solubilized from isolated plasma membranes. The kinetic properties of the enzyme are not markedly altered by its dissociation from the membrane matrix. However, there are significant differences in its behavior toward Mg2+ which suggest a structural role for Mg2+ in liver alkaline phosphatase.     

Fluorescence of cowpea-chlorotic-mottle virus modified with pyridoxal 5'-phosphate

Cowpea chlorotic mottle virus (CCMV), which is stable at pH 5.0, has been modified at this pH with 0.5--0.7 pyridoxal 5'-phosphate molecules per protein subunit. The fluorescence properties of the labelled CCMV protein in different aggregation states of the virus provide information about the labelled part of the protein and the changes induced in its environment, when the nucleo-protein particles are swollen or dissociated. Fluorescence excitation and emission spectra indicate the presence of radiationless energy transfer from the aromatic amino acid residues to the label. Comparison of the fluorescence lifetimes of the labelled and the unlabelled protein confirms the existence of energy transfer. The mobility of the labelled part, which can be estimated from the fluorescence polarization of pyridoxal phosphate chromophore, is higher than expected from the dimensions of the virus and the protein subunits. Polarization values and the fluorescence lifetimes depend on the presence of small amounts of NaCl or MgCl2 in the buffer solution at pH 7.5. This is due to structural changes in the vicinity of the pyridoxal phosphate label of the RNA and of the protein part.     

L-theanine elicits an umami taste with inosine 5'-monophosphate

We investigated the taste synergy between L-theanine and the flavour enhancer, inosine 5'-monophosphate (IMP), by using a human sensory evaluation. When L-theanine was added to IMP, only the umami taste was enhanced. We then investigated this synergistic effect of L-theanine in mice by gustatory nerve recording. We confirmed the synergism between L-theanine and IMP for the umami taste.     

5'-deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: studies with 5'-nucleotidase and alkaline phosphatase

The interaction of 5'-deoxy-5'-thioadenosine 5'-monophosphate (A(S)MP) and 5'-deoxy-5'-thioinosine 5'-monophosphate (I(S)MP) with snake venom, 5'nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5'-monophosphate and inosine 5'-monophosphate in which sulfur replaces oxygen as the bridge between the 5'-carbon of the ribose and the phosphorous. The P-S bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The Km for A(S)MP was 270 microM and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 microM and 530 nmol/min/mg protein, respectively. In contrast, 5'-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5'-nucleotidase hydrolysis of AMP and IMP, respectively, with Ki values of 975 and 13 microM. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the Ki for I(S)MP by 100-fold, to a value of 0.15 microM.