Biomedical subjects

Joshua J Woodward

Publications and source records attributed to Joshua J Woodward.

3 recordsLinked to original sources

PubMed2006

An Escherichia coli expression-based method for heme substitution.

Heme reconstitution with porphyrin analogs is a powerful approach toward understanding the molecular function of heme proteins; present methods, however, have not proven to be generally useful. Here we describe the development and application of an expression-based method for introducing modified porphyrins. The approach allows efficient incorporation of heme analogs using a widely available bacterial strain and offers an attractive alternative to present reconstitution methods that subject proteins to harsh, denaturing conditions.

Chromatography, High Pressure Liquid↗

PubMed2006

NG-hydroxyguanidines from primary amines.

A concise and general method for the preparation of N(G)-hydroxyguanidines from primary amines is reported. Using available and readily prepared materials, primary amines are converted to protected N(G)-hydroxyguanidines in a one-pot procedure followed by deprotection under nonreducing conditions. The method has been successfully applied to a number of examples including a high-yielding preparation of N(G)-hydroxy-L-arginine, the intermediate in the enzymatic conversion of L-arginine to nitric oxide and L-citrulline by nitric oxide synthase.

Amines↗

PubMed2004

Alanine racemase free energy profiles from global analyses of progress curves.

Free energy profiles for alanine racemase from Bacillus stearothermophilus have been determined at pH 6.9 and 8.9 from global analysis of racemization progress curves. This required a careful statistical design due to the problems in finding the global minimum in mean square for a system with eight adjustable parameters (i.e., the eight rate constants that describe the stepwise chemical mechanism). The free energy profiles obtained through these procedures are supported by independent experimental evidence: (1). steady-state kinetic constants, (2). solvent viscosity dependence, (3). spectral analysis of reaction intermediates, (4). equilibrium overshoots for progress curves measured in D(2)O, and (5). the magnitudes of calculated intrinsic kinetic isotope effects. The free energy profiles for the enzyme are compared to those of the uncatalyzed and the PLP catalyzed reactions. At pH 6.9, PLP lowers the free energy of activation for deprotonation by 8.4 kcal/mol, while the inclusion of apoenzyme along with PLP additionally lowers it by 11 kcal/mol.

Alanine↗