Activation of a covalent enzyme-substrate bond by noncovalent interaction with an effector.
The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon beta-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD(+), even where the "redox" properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H(2)O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native "active-thiol" furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the "two equivalent acyl sites," even though it eliminates the apparent "negative cooperativity" in NAD binding.
D000179:Acrylates; D000215:Acylation; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D005399:Fishes; D005663:Furans; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D005994:Glycerophosphates; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000179:Acrylates; D005663:Furans; D005994:Glycerophosphates; D008024:Ligands; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases