Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00790 (PGA)
 2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans which catalyzes the irreversible oxidation of D-glyceraldehyde-3 phosphate (D-G3P) into 3-phosphoglycerate (3-PGA) in the presence of NADP belongs to the aldehyde dehydrogenase (ALDH) superfamily. Oxidation of D-G3P into 3-PGA by GAPN involves the formation of a covalent enzyme intermediate via the nucleophilic attack of the invariant Cys-302. Titration of Cys-302 in the apo-enzyme by two different kinetic probes, iodoacetamide and 2,2'-dipyridyl disulfide, shows a pK(app) of 8.5 and a chemical reactivity surprisingly low compared to a reactive and accessible thiolate. Binding of NADP causes a strong increase of the reactivity of Cys-302-which is time dependent-with a pK(app) shift from 8.5 to 6.1. Concomitant with the increase in the Cys-302 reactivity, an additional protein fluorescence quenching is observed. These data suggest that cofactor binding induces at least a local conformational rearrangement within the active site. The efficiency of the rearrangement depends on the structure of the cofactors and on the protonation of an amino acid with a pK(app)( )()of 5.7. The rate of the rearrangement also strongly increases when temperature decreases. The data on the conformational rearrangement also reveal an amino acid with a pK(app) of 7.6 whose deprotonation increases the reactivity of the thiolate of Cys-302 by a 3-fold factor. The nature of the amino acid involved-which should be located close to Cys-302 in the holo-active form-is likely the invariant Glu-268. Changing Glu-268 into Ala or Cys-302 into Ala leads to mutants in which the rearrangement is only efficient in the presence of saturating concentrations of both NADP and G3P. The structural aspects of the conformational rearrangement occurring during the catalytic process in the wild-type GAPN should include at least reorientation of both Cys-302 and Glu-268 side chains and repositioning of the nicotinamide ring of the cofactor to permit the chemical activation of Cys-302 and the formation of an efficient ternary complex. Thus, it is likely that the conformation of the active site in the reported X-ray structures of ALDHs determined so far in the presence of cofactor, in which the side chains of Cys-302 and Glu-268 are 6.7 A apart from each other, does not represent the biological active form.
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PMID:Evidence for the chemical activation of essential cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans. 1050 67

Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was enriched 2600-fold by (NH(4))(2)SO(4) fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6-phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi(2-), Cl(-), SO(4) (2-), NO(3) (-), HAsO(4) (2-), and HCO(3) (-) but was not affected by NH(4) (+). Pi and Mg(2+) changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg(2+) was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3-PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P(2) had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light.
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PMID:Cytosolic phosphofructokinase from spinach leaves : I. Purification, characteristics, and regulation. 1666 57

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.
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PMID:Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells. 2290 79