Gerson F. Pinto

Pocket computer program for fitting the Michaelis-Menten equation

This paper presents a program written for the Radio Shack TRS-80 or the Sharp PC 1211 pocket computers for fitting the Michaelis-Menten equation to experimental data by a non-linear regression analysis. The program determines the kinetic parameters Km and Vm, their standard errors and the residual standard error. In addition it performs a simulation of a kinetic experiment using the parameter values previously determined.

Simultaneous enzymatic synthesis of (S)-3-fluoroalanine and (R)-3-fluorolactic acid

Abstract A coupled enzymatic system for the simultaneous synthesis of ( S )-3-fluoroalanine ( 1a ) and ( R )-3-fluorolactic acid ( 3 ) with l -alanine dehydrogenase ( l -AlaDH) from Bacillus subtilis and rabbit muscle l -lactate dehydrogenase ( l -LDH) using rac - 1 and NAD + is described. Analysis of isolated products of the laboratory preparative scale process revealed 1a in 60% yield and 88% ee and 3 in 80% yield and over 99% ee. The compounds 1a and 3 represent chiral building blocks for the synthesis of several products with pharmacological activity.

A microcomputer program for fitting enzyme inhibition rate equations.

This paper presents a computer program written in BASIC for fitting different enzyme inhibition kinetic models. The program, based on a nonlinear least-squares regression, can be run on any microcomputer with the CP/M operating system. Weighting of observed initial velocities is decided by the user by assessing constant variance, proportional variance or by incorporation of the variances calculated by a subroutine. The program also uses robust regression by bisquare weighting. All questions concerning data input, type of rate function, type of weight and the use of bisquare regression appear on the video display unit.

Resolution of dl-hydantoins by d-hydantoinase from Vigna angularis: Production of highly enantioenriched N-carbamoyl-d-phenylglycine at 100% conversion

Summary.d-Hydantoinase from Vigna angularis hydrolyzed rac-5-monosubstituted-hydantoins with polar and aromatic side chains and dihydrothymine but rac-5,5-disubstituted-hydantoins were not substrates of this enzyme. 5-Phenylhydantoin was the best substrate. By using this substrate, N-carbamoyl-d-phenylglycine was obtained in quantitative yield and over 98% ee.

Steady-state kinetic mechanism of Thermoanaerobium brockii alcohol dehydrogenase: A study of discrimination between alternative kinetic models

Two steady-state kinetic mechanisms have been proposed for the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase (TBADH): the sequential ordered BiBi mechanism and the Theorell-Chance BiBi mechanism. Discrimination between these kinetic mechanisms was achieved by analyzing the inhibition produced by acetone, the first product of the reaction sequence in relation to the second substrate of the enzyme (2-propanol). Two types of experimental data were used: a data set combining initial rate experiments in the absence of products with data involving inhibition by acetone in relation to 2-propanol as variable substrate; and, data sets just containing product inhibition experiments by acetone with 2-propanol as variable substrate. The results obtained strongly suggest that the kinetic mechanism of TBADH is described by the Theorell-Chance BiBi kinetic model.

Three different coupled enzymatic systems for in situ regeneration of NADPH

Three different coupled enzymatic systems used in the reduction of sulcatone by alcohol dehydrogenase from Thermoanaerobium brockii (TBADH), were kinetically compared. The first one involved the use of TBADH for both the principal and recycling reactions and 2-propanol 20%, v/v as the recycling substrate. The other two were based on the use of a different enzyme, glucose- or glucose-6-phosphate dehydrogenases, for in situ regeneration of NADPH. The coupled-substrate approach achieved 100% of conversion against 84% of the other two systems.

Kinetic mechanism of the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase

Abstract The reaction of oxidation of 2-propanol to acetone in the presence of NADP catalyzed by Thermoanaerobium brockii alcohol dehydrogenase (TBADH) has been studied for gaining insight into the coupled-substrate approach that has been extensively used for NADPH regeneration in preparative-scale reductions catalyzed by this enzyme. The data obtained in initial velocity experiments in the absence of products as well as those involving product inhibition and dead-end inhibition by pyrazole suggest that the reaction mechanism is of the Theorell-Chance BiBi type and in which the coenzymes bind to the free form of the enzyme. The seven kinetic parameters that characterize this kinetic model were estimated by non-linear regression analysis using the appropriate rate equations. The kinetic mechanism proposed in the present work is fully compatible with the coupled-substrate approach used for regeneration of NADPH in reduction reactions catalyzed by TBADH.

Kinetic aspects of the enantiospecific reduction of sodium 3-fluoropyruvate catalyzed by rabbit muscle L-lactate dehydrogenase : Production of homochiral (R)-3-fluorolactic acid methyl ester

Abstract In the present work the reduction of sodium 3-fluoropyruvate catalyzed by rabbit muscle l -lactate dehydrogenase ( l -LDH) was studied by means of initial rate experiments. Estimates of the limiting values of the kinetic parameters of the reaction were obtained. A kinetic mechanism involving a compulsory order of substrate binding to l -LDH, with NADH being the first substrate, is proposed. In addition, a simple procedure for the enantiospecific reduction of 3-fluoropyruvate catalyzed by this enzyme in a laboratory preparative scale is described. NADH was used in catalytic concentration by utilizing a NADH in situ regeneration system consisting of the oxidation of cis -1,2-bis(hydroxymethyl)cyclohexane (BHMC, 7 ) to chiral lactone (+)-(1 R , 6 S )- cis -8-oxabicyclo[4.3.0]nonan-7-one ( 8 ) catalyzed by horse liver alcohol dehydrogenase (HLADH). Analysis of the isolated product (100% conversion), after methylation, revealed the presence of ( R )-3-fluorolactic acid methyl ester as the unique product that was obtained in 80% overall yield and ee > 99%. This compound represents an important chiral building block for the synthesis of several products with biological and/or pharmacological activity.

Pocket computer program for fitting the Hill equation

This paper presents a program written in the BASIC language for the Radio Shack TRS-80 PC-1 or the Sharp PC 1211 pocket computers for fitting the Hill equation to experimental data. The program is based on a procedure involving a combination of an optimization technique and non-linear regression. The program determines precisely all three parameters of the Hill equation and in addition, it performs a simulation of the kinetic experiment using the parameter values previously estimated, calculating the sum of squares of residuals.

Enzymatic laboratory scale production of homochiral (R)-3-fluorolactic acid methyl ester via enantiospecific reduction of sodium fluoropyruvate catalyzed by rabbit muscle l-lactate dehydrogenase (l-LDH)

Abstract In the present work a simple laboratory procedure for the synthesis of ( R )-3-Fluorolactic Methyl Ester( 1 ) is described. A coupled enzymatic system formed by rabbit muscle l -lactate dehydrogenase ( l -LDH), horse liver alcohol dehydrogenase (HLADH), fluoropyruvic acid sodium salt, NAD + and cis-1,2-bis(hydroxymethyl)cyclohexane were found to be very effective for production of ( 1 ) in 80% overal yield and ee > 99%.