Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements

Biochemistry ◽  
1988 ◽  
Vol 27 (5) ◽  
pp. 1604-1610 ◽  
Author(s):  
Karen S. Anderson ◽  
James A. Sikorski ◽  
Kenneth A. Johnson
Keyword(s):  
Stopped Flow ◽  
Inhibitor Binding ◽  
Fluorescence Measurements ◽  
Phosphate Synthase

Synergistic Inhibitor Binding toStreptococcus pneumoniae5-Enolpyruvylshikimate-3-phosphate Synthase with Both Monovalent Cations and Substrate

Biochemistry ◽  
2000 ◽  
Vol 39 (33) ◽  
pp. 10140-10146 ◽  
Author(s):  
Wensheng Du ◽  
Wu-Schyong Liu ◽  
David J. Payne ◽  
Michael L. Doyle
Keyword(s):  
Monovalent Cations ◽  
Inhibitor Binding ◽  
Phosphate Synthase

scholarly journals Stopped-flow Fluorescence Studies of Inhibitor Binding to Tyrosinase fromStreptomyces antibioticus

2003 ◽  
Vol 279 (14) ◽  
pp. 13425-13434 ◽  
Author(s):  
Armand W. J. W. Tepper ◽  
Luigi Bubacco ◽  
Gerard W. Canters
Keyword(s):  
Stopped Flow ◽  
Inhibitor Binding ◽  
Fluorescence Studies

scholarly journals Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

1979 ◽  
Vol 177 (2) ◽  
pp. 697-705 ◽  
Author(s):  
Karen S. McGurk ◽  
H. Olin Spivey
Keyword(s):  
Secondary Structure ◽  
Pyruvate Carboxylase ◽  
Conformational Transitions ◽  
Sulphonic Acid ◽  
Chicken Liver ◽  
Stopped Flow ◽  
Acetyl Coa ◽  
Allosteric Effector ◽  
Fluorescence Measurements ◽  
Secondary Structure Composition

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO3 and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10–13%; ATP, 21–24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO3 had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the ε-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499–3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% β-pleated sheet and 66% other structural types.

scholarly journals Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 629-637 ◽  
Author(s):  
F. Mark Dickinson ◽  
Christopher J. Dickenson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Catalytic Mechanism ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Stopped Flow ◽  
Phenol Red ◽  
Yeast Alcohol Dehydrogenase ◽  
Fluorescence Measurements ◽  
High Concentrations

Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD+ in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pKa approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185–4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD+ release from the enzyme–NAD+–ethanol product complex. This finding permits calculation of K3, the dissociation constant for ethanol from the enzyme–NAD+–ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613–627). The calculations show that K3 varies very little with pH in the range 5.95–8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme–NADH–acetaldehyde]/ [enzyme–NAD+–ethanol] at equilibrium. The values obtained were in the range 0.11±0.04, and this value together with estimates of K3 was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.

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