Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity

Abstract

The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining. The pyridoxal form of E. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL

“…Protein concentrations were determined on the basis of values of absorbance at 278 nm. The theoretical molar extinction coefficients for the proteins were calculated using previously described method (51).…”

Free Energy Requirement for Domain Movement of an Enzyme

“…Protein Purification and Crystallization-AspAT was purified as described previously (11). It was then converted to the apoenzyme by adding 10 mM cysteine sulfinate and 0.5 M potassium phosphate (pH 8.0); excess PLP and cysteine sulfinate were removed using Sephadex G-50 gel filtration.…”

“…The structures were determined by molecular replacement methods using the structure of the PLP form (PDB code 1ARS) or the PMP form (PDB code 1AMQ) as the starting model. Model refinement was performed by the CCP4 program suite (21) version 3.51, X-PLOR (22) Kinetic Analysis-The half-transamination reactions of the PLP form of the enzymes were followed spectrophotometrically at 360 nm as described previously (11). The reaction conditions used were 50 mM HEPES, 100 mM KCl, and 10 M EDTA, pH 8.0, at 25°C.…”

Structure of the stand-alone RAM-domain protein fromThermus thermophilusHB8

“…The SraA protein was concentrated with a Vivaspin 20 concentrator (5 kDa molecular-weight cutoff, Sartorius, AG, Goettingen, Germany). The protein concentration was determined by measuring the absorbance at 280 nm (Kuramitsu et al, 1990).…”