irreversible inhibitor, a saccharin derivative, reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP, covalent inhibitor mechanism, overview. Inhibition of LmPYK by the compound is time-dependent
the nitrogen analogue N-(2,5-dimethylphenyl)-1,2-benzothiazol-3-amine 1,1-dioxide of 3-(2,5-dimethylphenoxy)-1,2-benzothiazole 1,1-dioxide is not inhibitory
the enzyme uses a rock and lock model allosteric mechanism, intersubunit interactions on the A-A and C-C interfaces strongly influence the allosteric effect whereas mutations affecting the intrasubunit A-C interface are less sensitive, overview. Conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control
the transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6o rigid body rocking motion of the A- and C-domain cores in each of the four subunits. Eight essential salt bridge locks form across the C-C interface providing tetramer rigidity with a coupled 7fold increase in reaction rate
the transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6o rigid body rocking motion of the A- and C-domain cores in each of the four subunits. Eight essential salt bridge locks form across the C-C interface providing tetramer rigidity with a coupled 7fold increase in reaction rate
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystals grown with ammonium sulfate as precipitant adopt an active-like conformation, with sulfate ions at the active and effector sites. Crystal soaking in sulfate-free buffers induces major conformational changes in the tetramer. The unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening of the tetramer caused by lateral movement of the C-domains
purified recombinant wild-type and mutant enzymes free and in complex with ligands ATP, oxalate, and fructose 2,6-bisphosphate, hanging drop vapour diffusion method, 0.0015 ml of 15 mg/ml protein in 20 mM TEA, pH 7.2, are mixed with 0.0015 ml of well solution composed of 10-16% PEG 8000, 20 mM TEA, pH 7.2, 50 mM MgCl2, 100 mM KCl, and 10-15% glycerol, 4°C or 17°C, 1week, X-ray diffraction structure determination and analysis
enzyme complexed with inhibitor 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid, mxing of 0.0015 ml of 10 mg/ml protein in 20 mM TEA, pH 7.2, 1 mM 1,3,6,8-pyrenetetrasulfonic acid, and 9 mM inhibitor, with 0.0015 ml of well solution containing 12-16% PEG 8000, 20 mM TEA buffer, pH 7.2, 50 mM magnesium chloride, 100 mM potassium chloride, and 10% glycerol, 1-2 days, and equilibration for 14 h over a well solution composed of 14-18% PEG 8,000, 20 mM TEA buffer, pH 7.2, 50 mM magnesium chloride, 100 mM potassium chloride, and 25% glycerol, X-ray diffraction structure determination and analysis at 2.65 A resolution