The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addi...

A microsomal deacetylase that catalyses the deacetylation of the O-glucoside of N-hydroxyacetanilide (GHA) was purified from guinea-pig liver. The activity was located exclusively in the microsomes and not detected in the cytosol. The purified GHA deacetylase was a trimeric protein with a molecular mass of 160+/-10 (S.D.) kDa composed of subunits of 53+/-2 kDa; its pI was 4.7. The N-terminal am...

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evolution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor ofMgATP in the MgATPinduced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K' = 20,UM was determined for MgADP. The release o...

Yeast chorismate mutase (EC 5.4.99.5) shows homotropic activation by the substrate, allosteric activation by tryptophan, and allosteric inhibition by tyrosine. In this study mutants of chorismate mutase have been found that remain sensitive to one allosteric effector (tryptophan) but insensitive to the other (tyrosine). These mutations are located in the catalytic domain: loop 220s (212-226) an...

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleava...

Cytochrome P-450 (P-450) 3A4 is the most abundant P-450 expressed in human liver and small intestine. P-450 3A4 contributes to the metabolism of approximately half the drugs in use today, and variations in its catalytic activity are important in issues of bioavailability and drug-drug interactions. The gene is known to be inducible by barbiturates, glucocorticoids, and rifampicin in humans and ...

Site-directed mutagenesis has been used to replace tyrosine-240 with phenylalanine in each of the catalytic chains of aspartate carbamoyltransferase. Tyrosine-240 is part of a loop in the structure of the enzyme, between residues 230 and 245, which undergoes a substantial conformation change as the enzyme becomes ligated [Krause, K. L., Volz, K. W. & Lipscomb, W. N. (1985) Proc. Natl. Acad. Sci...

Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids, and other ligands. In addition, CaSR is a pleiotropic receptor regulating several...

The structural basis of cooperativity of progesterone hydroxylation catalyzed by human cytochrome P450 3A4 has been investigated. A recent study suggested that substitution of larger side chains at positions Leu-211 and Asp-214 partially mimics the action of effector by reducing the size of the active site. Based on predictions from molecular modeling that Phe-304 in the highly conserved I heli...