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Biomedical subjects

A S Mildvan

Publications and source records attributed to A S Mildvan.

At least 127 records · Page 7Linked to original sources
PubMed1981

Conformations and arrangement of substrates at active sites of ATP-utilizing enzymes.

The phosphoryl transferring enzymes pyruvate kinase, cAMP-dependent protein kinase and the pyrophosphoryl transferring enzyme PP-Rib-P synthetase utilize the beta, gamma bidentate metal--ATP chelate (delta-isomer) as substrate, as determined with substitution-insert CrIIIATP or CoIII(NH3)4ATP complexes. In addition, these enzymes bind a second divalent cation, which is an essential activator for pyruvate kinase and PP-Rib-P synthetase and an inhibitor of protein kinase. The enzyme-bound metal has been used as a paramagnetic reference point in T1 measurements to determine distances to the protons and phosphorus atoms of the bound nucleotide and acceptor substrates. These distances have been used to construct models of the conformations of the bound substrates. The activating metal forms a second sphere complex of the metal-nucleotide substrate on pyruvate kinase and PP-Rib-P synthetase while the inhibitory metal directly coordinates the polyphosphate chain of the metal-nucleotide substrate on protein kinase. Essentially no change is found in the dihedral angle at the glycosidic bond of ATP upon binding to pyruvate kinase (chi = 30 degrees), an enzyme of low base specificity, but significant changes in the torsional angle of ATP occur on binding to protein kinase (chi = 84 degrees) and PP-Rib-P synthetase (chi = 62 degrees), enzymes with high adenine-base specificity. Intersubstrate distances, measured with tridentate CrATP or beta, gamma bidentate CrAMPPCP as paramagnetic reference points, have been used to deduce the distance along the reaction coordinate on each enzyme. The reaction coordinate distances on pyruvate kinase (# +/- 1 A) and PP-Rib-P synthetase (not less than 3.8 A) are consistent with associative mechanisms, while that on protein kinase (5 +/- 0.7 A) allows room for a dissociative mechanism.

Adenosine Triphosphate↗
PubMed1981

Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis.

The contribution of proofreading to the fidelity of catalysis by DNA polymerases has been determined with deoxyribonucleoside [1-thio]triphosphate substrates. These analogues, which contain a sulfur in place of an oxygen on the alpha phosphorus, are incorporated into DNA by DNA polymerases at rates similar to those of the corresponding unmodified deoxynucleoside triphosphates. The fidelity of DNA synthesis was measured with phi X174 am3 DNA; reversion to wild type occurs most frequently by a single base substitution, a C for a T at position 587. By using avian myeloblastosis virus DNA polymerase and DNA polymerase beta (enzymes without a proofreading 3' leads to 5' exonucleolytic activity), substitution of deoxycytidine thiotriphosphate in the reaction mixture did not alter fidelity. In contrast, with DNA polymerases from E. coli (DNA polymerase I) and bacteriophage T4 (enzymes containing a proofreading activity), fidelity was markedly reduced with deoxycytidine [1-thio]triphosphate. DNA containing phosphorothioate nucleotides is insensitive to hydrolysis by the exonuclease associated with these prokaryotic DNA polymerases. These combined results indicate that the deoxynucleoside [1-thio]triphosphates have normal base-pairing properties; however, once misinserted by a polymerase, they are not excised by proofreading. Proofreading of a C:A mismatch at position 587 is thereby found to contribute 20-fold to the fidelity of E. coli DNA polymerase I and a greater amount to the fidelity of bacteriophage T4 DNA polymerase.

Avian Myeloblastosis Virus↗
PubMed1980

Magnetic resonance measurements of intersubstrate distances at the active site of protein kinase using substitution-inert cobalt(III) and chromium(III) complexes of adenosine 5'-(beta, gamma-methylenetriphosphate).

Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.

Adenosine Triphosphate↗
PubMed1980

Presence of a flavin semiquinone in methanol oxidase.

Methanol oxidase from Hansenula polymorpha contains five "red" flavin semiquinones and two oxidized flavins per octamer. Addition of substrate results in the reduction of the two oxidized flavins but does not affect the flavin semiquinones. Enhanced water proton relaxation rates indicate that the unpaired electron of the flavin semiquinones is accessible to the solvent and this accessibility is significantly decreased upon binding of the suicide inhibitor cyclopropanol. In the native enzyme, the semiquinones are not oxidizable by air. All flavins were resolved from the enzyme, and holoenzyme was reconstituted by addition of oxidized flavin. The reconstituted enzyme was catalytically active. The specific activity was 50% that of the original enzyme. It was concluded that the semiquinone is not required for the oxidation of methanol, although it may be present at an otherwise intact site.

Alcohol Oxidoreductases↗