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At least 307 records · Page 17Linked to original sources
PubMed1996

Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and some bacteria. The enzyme belongs to a large family of enzymes that use pyridoxal 5'-phosphate as an essential cofactor. We previously analyzed the informational content contained in each residue of a conserved glycine loop, which we proposed to form part of the cofactor binding site [Gong, J., & Ferreira, G. C. (1995) Biochemistry 34, 1678-1685]. We found that Gly-142 and -144 contain high informational content, and we identified G144A, G144S, G144T, and G142C as functional mutants. Here, the catalytic parameters, cofactor affinities, and spectral and thermostability properties of these four glycine mutants are determined to examine the function of the glycine loop. In addition, computer models of the glycine loops from the wild-type and mutant enzymes were generated, using glycogen phosphorylase b as the structural template. G144A, G144S, G144T, and G142C displayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and 15-fold increases, respectively, in the dissociation constant value for binding of the cofactor. While the turnover numbers for G144A, G144S, G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value, respectively, the K(m) values for both substrates remained unchanged, with the exception of the G142C K(m)Gly, which showed a 4-fold increase. The UV-visible and CD spectra of Gly-144 mutants were similar to those of the wild type; however, the spectral properties of G142C suggest that this mutant binds the cofactor in a different mode at the active site. G144A, G144S, G144T, and G142C were also found to be less stable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 3.5, 3, 3.5, and 5 degrees C, respectively, lower than that of the wild-type enzyme. Collectively, computer modeling of the wild-type and mutant forms of the ALAS glycine loop and biochemical and spectroscopic characterization of G144A, G144S, G144T, and G142C strongly suggest that the conserved glycine loop in 5-aminolevulinate synthase is a pyridoxal 5'-phosphate cofactor binding motif.

5-Aminolevulinate Synthetase↗
PubMed1995

Hyperhomocysteinemia and low pyridoxal phosphate. Common and independent reversible risk factors for coronary artery disease.

BACKGROUND: High plasma homocysteine is associated with premature coronary artery disease in men, but the threshold concentration defining this risk and its importance in women and the elderly are unknown. Furthermore, although low B vitamin status increases homocysteine, the link between these vitamins and coronary disease is unclear. METHODS AND RESULTS: We compared 304 patients with coronary disease with 231 control subjects. Risk factors and concentrations of plasma homocysteine, folate, vitamin B12, and pyridoxal 5'-phosphate were documented. A homocysteine concentration of 14 mumol/L conferred an odds ratio of coronary disease of 4.8 (P < .001), and 5-mumol/L increments across the range of homocysteine conferred an odds ratio of 2.4 (P < .001). Odds ratios of 3.5 in women and of 2.9 in those 65 years or older were seen (P < .05). Homocysteine correlated negatively with all vitamins. Low pyridoxal 5'-phosphate (< 20 nmol/L) was seen in 10% of patients but in only 2% of control subjects (P < .01), yielding an odds ratio of coronary disease adjusted for all risk factors, including high homocysteine, of 4.3 (P < .05). CONCLUSIONS: Within the range currently considered to be normal, the risk for coronary disease rises with increasing plasma homocysteine regardless of age and sex, with no threshold effect. In addition to a link with homocysteine, low pyridoxal-5'-phosphate confers an independent risk for coronary artery disease.

Age Factors↗
PubMed1996

Characterization of pyridoxal phosphate as an optical label for measuring electrostatic potentials in proteins.

This paper presents data which allow one to characterize pyridoxal 5'-phosphate (PLP) as an optical label for electrostatic potential measurements in proteins. Experimental studies were carried out with 6-N-(5'-phosphopyridoxyl)-2-N-acetyl-lysyl methyl ester (PLP-ALME) as a model compound which simulates PLP covalently bound to a protein. Calculations of electrostatic potential maps were done using the model compound 2,4-dimethyl-3-hydroxy-5-hydroxymethyl phosphate-pyridine (DHHPP). Studies on relative changes in PLP-ALME fluorescence and absorbance vs. pH resulted in four pKa values of PLP-ALME in solution which can be used as intrinsic pKa values (pKint) of the ionizable groups of the label covalently bound to the protein. The pKa values obtained from fluorescence data are 4.1, 5.6, 8.7 and 11.1 and those from absorbance data are 3.1, 4.7, 8.7 and 11.0. The differences between corresponding pKa values are related to differences in PLP-ALME behaviour between the excited and ground electronic states and to intramolecular charge-charge interactions. Quenching of PLP-ALME fluorescence by I-, acrylamide and Cs+ at pH 6, 7 and 8 shows that in the case of I- and acrylamide the Stern-Volmer constants of quenching (Ksv) decrease with increasing pH, while the opposite is true for Ksv of Cs+. These results as well as the analysis of electrostatic potential maps of DHHPP show that with its ampholytic character PLP can be used to measure local electrostatic potentials in the pH range 5-9.

Acrylamide↗
PubMed1982

Effect of different levels of vitamin B6 in the diet of rats on the content of pyridoxamine-5'-phosphate and pyridoxal-5'-phosphate in the liver.

The pyridoxal-5'-phosphate (PLP) and pyridoxamine-5'-phosphate (PMP) contents were determined in livers of rats fed diets containing different levels of vitamin B6 (0.3-22.9 ppm). Maximum growth was attained at vitamin B6 intake levels of greater than or equal to 24 micrograms/day. Maximum PLP and PMP levels in a crude liver homogenate were reached at intake levels of 75 and 9 micrograms/day respectively. In the 100 000 g supernatant fraction of liver the maximum PLP level was reached at intake levels of greater than or equal to 37 micrograms/day. The PLP/PMP-ratio of the crude liver homogenate increased from 0.6 up to values of appr. 1.5 at the higher intake levels. The PLP/PMP-ratio in the 100 000 g supernatant was more variable and did not show a consistent trend at varying vitamin B6 intakes.

Animals↗
PubMed1985

Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli.

NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics. The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol. The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent. In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.

Escherichia coli↗
PubMed1985

Two forms of rat brain glutamic acid decarboxylase differ in their dependence on free pyridoxal phosphate.

There are two forms of glutamate decarboxylase (GAD) found in the rat brain. One form (form A) does not require exogenous pyridoxal-5'-phosphate (PLP) for activity whereas another form (form B) requires exogenous PLP for activity. These two forms differ greatly in temperature sensitivity, inactivation, and reactivation by the removal and readdition of PLP, electrophoretic mobility, and regional distribution. For instance, forms A and B are inactivated to an extent of 91% and 10%, respectively, by the treatment at 45 degrees C for 30 min; form A is greatly inactivated (77%) by the removal of PLP by aminooxyacetic acid and the readdition of PLP, whereas form B is only slightly inactivated (7%). Forms A and B can be clearly separated by 5% polyacrylamide gel electrophoresis in which form A migrates faster than form B. In all 10 brain regions studied, form A is present in smaller amounts than form B. This difference is greatest in the superior colliculus (the ratio of B to A is about 5), while in the locus coeruleus and cerebellum, forms A and B are present in nearly equal proportion. Forms A and B are similar with respect to relative abundance in hypotonic, isotonic, and hypertonic preparations, inhibition of catalytic activity by a carbonyl-trapping agent, immunochemical properties, and chromatographic patterns in a variety of systems. The significance of forms A and B and PLP in the regulation of gamma-amino-butyric acid (GABA) level is also discussed.

Aminooxyacetic Acid↗
PubMed1980

Pyridoxal phosphate as a probe of reovirus transcriptase.

The ribonucleoprotein core of reovirus is a multienzyme complex that transcribes messenger ribonucleic acid (mRNA) from double-stranded RNA templates. So far, the core has resisted attempts to disassemble it and identify the polypeptide species responsible for RNA polymerase activity. As an alternative approach, we tested pyridoxal 5-phosphate (PLP) as a potential affinity labeling reagent for reovirus transcriptase in vitro; PLP has been used as an affinity reagent for cellular and viral nucleic acid polymerases. We found that PLP inhibited reovirus transcriptase reversibly (apparent Ki = 0.2 mM), but the inhibition was noncompetitive with respect to each of the four ribonucleoside triphosphates. This interaction required both the aldehyde and phosphate moieties in PLP, since pyridoxamine and pyridoxal were relatively inactive. To identify the polypeptides involved, we labeled the PLP--core complex by reductive alkylation with [3H]borohydride. At PLP concentrations close to the apparent Ki, labeling was selective for the two largest virion polypeptides, lambda 1 and lambda 2. At saturation, there were only 10 high-affinity PLP binding sites per core in each of the lambda polypeptide species. These findings implicate either or both lambda polypeptide species in viral transcription and they indicate that a special population, representing no more than 10% of the total lambda molecules in each core, participates in RNA synthesis.

Borohydrides↗
PubMed2001

Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme.

The established pathways from serine to ethanolamine are indirect and involve decarboxylation of phosphatidylserine. Here we show that plants can decarboxylate serine directly. Using a radioassay based on ethanolamine (Etn) formation, pyridoxal 5'-phosphate-dependent l-serine decarboxylase (SDC) activity was readily detected in soluble extracts from leaves of diverse species, including spinach, Arabidopsis, and rapeseed. A putative Arabidopsis SDC cDNA was identified by searching GenBank for sequences homologous to other amino acid decarboxylases and shown by expression in Escherichia coli to encode a soluble protein with SDC activity. This cDNA was further authenticated by complementing the Etn requirement of a yeast psd1 psd2 mutant. In a parallel approach, a cDNA was isolated from a rapeseed library by its ability to complement the Etn requirement of a yeast cho1 mutant and shown by expression in E. coli to specify SDC. The deduced Arabidopsis and rapeseed SDC polypeptides are 90% identical, lack obvious targeting signals, and belong to amino acid decarboxylase group II. Recombinant Arabidopsis SDC was shown to exist as a tetramer and to contain pyridoxal 5'-phosphate. It does not attack d-serine, l-phosphoserine, other l-amino acids, or phosphatidylserine and is not inhibited by Etn, choline, or their phosphoesters. As a soluble, pyridoxal 5'-phosphate enzyme, SDC contrasts sharply with phosphatidylserine decarboxylases, which are membrane proteins that have a pyruvoyl cofactor.

Amino Acid Sequence↗