Biomedical subjects

N F Phillips

Publications and source records attributed to N F Phillips.

22 records · Page 2Linked to original sources

PubMed1988

Involvement and identification of a tryptophanyl residue at the pyruvate binding site of transcarboxylase.

Transcarboxylase (TC) from Propionibacterium shermanii consists of a central hexameric 12S subunit to which 6 outer dimeric 5S subunits are attached through 12 biotinyl 1.3S subunits. The enzyme catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate, forming oxalacetate and propionyl-CoA. The pyruvate binding site, located on the 5S subunit, was examined by monitoring the intrinsic fluorescence quenching accompanying the incremental addition of pyruvate to either TC or the 5S subunit. The quenching studies indicate that there are two binding sites for pyruvate with apparent dissociation constants of 0.23 and 1.25 mM for intact TC and of 0.18 and 1.20 mM for the outer 5S subunit. The microenvironment of the Trp(s) sensitive to pyruvate binding was analyzed by using the neutral quencher acrylamide. With TC, the fractional accessible fluorescence (fa) was 0.64, whereas a fa value of 0.56 was obtained in the presence of pyruvate. A 27% decrease in fa was observed with the outer 5S subunit in the presence of pyruvate as compared to the free 5S subunit. By labeling the outer subunit in the absence of pyruvate with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), a tryptic peptide containing DNPS-labeled Trp was isolated; the sequence was determined and identified with the amino-terminal residues 67-75 of the outer subunit that has been derived from DNA-sequencing studies. Trp-73 contained the DNPS label; its labeling was inhibited by pyruvate. A sequence comparison with other biotinyl enzymes shows that the sequence 67-75 is highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗

PubMed1988

The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate.

Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.

Adenosine Monophosphate↗

PubMed1986

Isolation of pyrophosphohistidine from pyrophosphorylated pyruvate, phosphate dikinase.

The pyrophosphoryl form of pyruvate, phosphate dikinase was prepared by incubation with adenosine 5'-[gamma-32P]triphosphate and isolated by gel chromatography. Previously a phosphorylated moiety had been isolated from the enzyme and was shown to be bound through a phosphoramidate linkage to the 3' nitrogen of a histidine residue [Spronk, A. M., Yoshida, H., & Wood, H. G. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4415]. This histidine residue has been considered to be the pyrophosphoryl and phosphoryl carrier between the three subsites of this enzyme. Previous attempts to isolate the putative [32P]pyrophosphohistidine have been unsuccessful due to the lability of the [32P]pyrophosphoryl-enzyme. By stabilization of the [32P]pyrophosphoryl-enzyme with diazomethane, it has been possible to isolate a [32P]-pyrophosphohistidine from the hydrolysates. To our knowledge this work constitutes the first direct demonstration of a pyrophosphorylated histidyl residue in an enzyme.

Adenosine Triphosphate↗

PubMed1983

Modification of pyruvate, phosphate dikinase with pyridoxal 5'-phosphate: evidence for a catalytically critical lysine residue.

Pyruvate, phosphate dikinase from Bacteroides symbiosus is strongly inhibited by low concentrations of pyridoxal 5'-phosphate. The inactivation follows pseudo-first-order kinetics over an inhibitor concentration range of 0.1-2 mM. The inactivation is highly specific since pyridoxine and pyridoxamine 5'-phosphate, analogues of pyridoxal 5'-phosphate, which lack an aldehyde group, caused little or no inhibition even at high concentrations. The unreduced dikinase-pyridoxal 5'-phosphate complex displays an absorption maxima near 420 nm, typical for Schiff base formation. Following reduction of the Schiff base with sodium borohydride, N6-pyridoxyllysine was identified in the acid hydrolysate. When the enzyme was incubated in the presence of pyridoxal 5'-phosphate and reducing agent, the ATP/AMP, Pi/PPi, and pyruvate/phosphoenolpyruvate isotopic exchange reactions were inhibited to approximately the same extent, suggesting that the modification of the lysyl moiety causes changes in the enzyme that affect the reactivity of the pivotal histidyl residue. Phosphorylation of the histidyl group appears to prevent the inhibitor from attacking the lysine residue. On the other hand, addition of pyridoxal 5'-phosphate to the pyrophosphorylated enzyme promotes release of the pyrophosphate and yields the free enzyme which is subject to inhibition.

Bacteroides↗