Crystallization of the FAD-independent acetolactate synthase of Klebsiella pneumoniae

Leucine and valine are formed in a common pathway from pyruvate in which the first intermediate is 2-acetolactate. In some bacteria, this compound also has a catabolic fate as the starting point for the butanediol fermentation. The enzyme (EC 4.1.3.18) that forms 2-acetolactate is known as either acetohydroxyacid synthase (AHAS) or acetolactate synthase (ALS), with the latter name preferred for the catabolic enzyme. A significant difference between AHAS and ALS is that the former requires FAD for catalytic activity, although the reason for this requirement is not well understood. Both enzymes require the cofactor thiamine diphosphate. Here, the crystallization and preliminary X-ray diffraction analysis of the Klebsiella pneumoniae ALS is reported. Data to 2.6 Angstrom resolution have been collected at 100 K using a rotating-anode generator and an R-AXIS IV++ detector. Crystals have unit-cell parameters a = 137.4, b = 143.9, c = 134.4 Angstrom, alpha = 90, beta = 108.4, gamma = 90degrees and belong to space group C2. Preliminary analysis indicates that there are four monomers located in each asymmetric unit

Crystallization of the catalytic subunit of Saccharomyces cerevisiae acetohydroyacid synthase

Acetohydroxyacid synthase (AHAS; E.C. 4.1.3.18) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids isoleucine, leucine and valine. It is a thiamin diphosphate-dependent enzyme which catalyses the decarboxylation of pyruvate and its condensation with either 2-ketobutyrate or a second molecule of pyruvate to give 2-aceto-2-hydroxybutyrate or 2-acetolactate, respectively. The enzyme is the target of sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Saccharomyces cerevisiae AHAS is reported. Data to 2.7 Å resolution have been collected using synchrotron radiation (Advanced Photon Source, Chicago). Crystals have unit-cell parameters a = 95.8, b = 110.0, c = 178.9 Å and belong to the space group P222. Preliminary analysis indicates there is one dimer located in each asymmetric unit

A computer program in BASIC for calculating gradients used in chromatography, electrophoresis and centrifugation

Concentration gradients are often used in separations based on chromatographic, electrophoretic and centrifugal methods. In this report, a BASIC computer program for calculating and graphically representing gradients is described. This GRADIENT program is intended to be run on IBM-compatible computers

Calculation of the molecular weight of proteins from electrophoretic and gel exclusion chromatographic experiments

The molecular weights of proteins or protein subunits are frequently estimated from the electrophoretic mobility in the presence of sodium dodecyl sulphate, or from the elution volume in gel filtration chromatography. Using a series of standards of known size, a calibration curve is prepared which often yields a straight line in a semilogarithmic plot; interpolation from this line then gives the size of the sample. While this method is simple to apply, it does not give any estimate of the accuracy of the determined size. Furthermore there is no objective criterion for deciding if the calibration curve is linear, or a procedure for dealing with non-linear calibration curves. Here I report a method and a computer program which uses appropriate statistical methods to select the best straight or curved calibration line. The size of the unknown is then interpolated and an estimate or the error in this value is calculated

Identification of an acetolactate synthase small subunit gene in two eukaryotes

Acetolactate synthase catalyses the first step in branched-chain amino acid biosynthesis. The bacterial enzyme contains two large and two small subunits but there is only limited and circumstantial evidence for a small subunit in the eukaryotic enzyme. Here this evidence is summarised and protein sequences of two putative eukaryotic small subunits, from a yeast and a red alga, are presented

Regression analysis of nonlinear Arrhenius plots: An empirical model and a computer program

The rates of most physical, chemical and biological processes vary with temperature and numerous instances have been reported in which Arrhenius plots of the experimental data appear to consist of two straight line segments joined by a relatively sharp break. An empirical model, based on a general hyperbola, is shown to be applicable to such systems; a nonlinear regression program is described which facilitates fitting of this function to experimental data which show either broad or sharp transitions. The program yields best estimates and standard errors of the temperature and ordinate value at the transition, and the two activation energies

Determination of inhibition constants, I50 values and the type of inhibition for enzyme-catalyzed reactions

A procedure is proposed for determining whether an inhibitor of an enzyme-catalyzed reaction is competitive, noncompetitive, or uncompetitive with respect to the substrate. The method is based on fitting the equation for noncompetitive inhibition to data obtained by measuring the rate of the reaction over a range of substrate and inhibitor concentrations. The results of this fit may suggest that the inhibition may be either competitive or uncompetitive, whereupon the data are reanalyzed using the appropriate equation. Comparison of this second fit with the first using an F test permits a statistical decision to be made on the type of inhibition. The chosen fit yields values and standard errors for the Michaelis-Menten parameters (maximum velocity and Michaelis constant), as well as the inhibition constant(s). From these values it is then possible to predict the I50, and its standard error, at any chosen substrate concentration, thereby facilitating comparison with results obtained with similar inhibitors, for homologous enzymes, or in other laboratories

Preparation of concentrated solutions of some common solutes by means of calculated molalities

The preparation of high molar concentrations of many solutes can often be very time-consuming owing to the substantial volume, and sometimes temperature, changes which occur as the solute dissolves. By contrast, the preparation of solutions of a given molality is much simpler since all that is required is to mix known weights of the solute and solvent. This paper describes a simple method and a computer program (MOLAL) for preparing known volumes of a given molarity for twenty commonly-used solutes, by means of calculated molalities; the density of the resulting solutions is also given