Browsing by Author "HINRICHS, MV"

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    AFFINITY LABELING OF RABBIT MUSCLE PYRUVATE-KINASE WITH DIALDEHYDE-ADP
    (1982) HINRICHS, MV; EYZAGUIRRE, J
    Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, 1 molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14C]dialdehyde-ADP into the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues/subunit react with the modifier, 2 of them being essential for activity. Dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase, the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures; and the enzyme possesses 2 modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss of activity.
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    ISOLATION AND SEQUENCE DETERMINATION OF AN ACTIVE-SITE PEPTIDE OF RABBIT MUSCLE PYRUVATE-KINASE
    (1987) BEZARES, G; EYZAGUIRRE, J; HINRICHS, MV; HEINRIKSON, RL; REARDON, I; KEMP, RG; LATSHAW, SP; BAZAES, S
    Rabbit muscle pyruvate kinase was inactivated by 2'',3''-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.
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    PYRUVATE-KINASE - STUDIES ON AFFINITY LABELING AND ACTIVE-SITE STRUCTURE USING THE RABBIT MUSCLE ENZYME
    (1985) EYZAGUIRRE, J; BAZAES, S; BEZARES, G; HINRICHS, MV; HEINRIKSON, RL; REARDON, I