Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies.

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.

Dynamics and interactions of the anion channel in intact human erythrocytes: an electron paramagnetic resonance spectroscopic study employing a new membrane-impermeant bifunctional spin-label.

We have developed a new membrane-impermeant, bifunctional spin-labeling reagent, bis-(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), and employed it in an electron paramagnetic resonance (EPR) study of the rotational diffusion of the anion-exchange channel (band 3) in intact human erythrocytes. BSSDP reacts in a covalent manner and with high specificity with the extracytoplasmic domain of band 3, forming a complex in which the spin-label is immobilized on the protein. The linear EPR spectrum of BSSDP-labeled intact erythrocytes is characteristic of a highly immobilized, spatially isolated nitroxide probe. The saturation-transfer EPR spectrum of the same sample indicates that the anion channel in intact erythrocytes exhibits rotational dynamics in the 0.1-1 ms correlation time range at 20 degrees C. Rotational dynamics in this motional domain are consistent with a strong interaction of the anion-exchange channel with the erythrocyte cytoskeleton. The saturation-transfer EPR spectrum of ghosts prepared from BSSDP-labeled erythrocytes indicates a significant increase in rotational mobility of the anion channel, suggesting a significant disruption on lysis of interactions between the anion channel and the cytoskeleton.

An electron-electron double-resonance study of interactions between [14N]- and [15N]stearic acid spin-label pairs: lateral diffusion and vertical fluctuations in dimyristoylphosphatidylcholine.

Vertical fluctuations of the terminal methyl groups of stearic acid acyl chains toward the surface of dimyristoylphosphatidylcholine (DMPC) bilayers have been investigated by using spin-label electron-electron double-resonance ( ELDOR ) methodology. Spin-label pairs consisting of two populations of stearic acid spin-labels were employed, each at 0.25 mol% concentration, where the nitroxides of the first population were 15N substituted and the nitroxides of the second contained 14N. Various combinations of labels with the nitroxide moieties located at carbons 5, 12, or 16 (C5, C12, C16) were used. ELDOR permits measurement of collision frequencies between the two constituents of the pair, for example, between 15N spin-labels at C5 and 14N labels at C16. Intramolecular contributions to the ELDOR effect including nitrogen nuclear relaxation are eliminated by the use of spin-label pairs. Above the main phase transition temperature, bimolecular collisions between C5 and C16 occur with about half the frequency of C16:C16 collisions. It is concluded that vertical fluctuations are very pronounced. A dependence of these fluctuations on temperature and pH has been observed. Lateral diffusion constants calculated from the bimolecular collision frequencies of C16:C16 pairs are 4.56 X 10(-8), 5.77 X 10(-8), and 8.09 X 10(-8) cm2/s at 27, 37, and 47 degrees C. These values are in good agreement with previous measurements of lipid diffusion in DMPC.

6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase from rat liver.

Rat liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated by [gamma-32P]ATP plus fructose-6-P or [2-32P]fructose-2,6-P2. The radioactivity co-migrated with homogeneous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase during sodium dodecyl sulfate-disc gel electrophoresis and the phosphoenzyme was acid-labile and base-stable. Hydrolysis of the phosphate group from phosphoenzyme prepared with either donor and the hydrolysis of their tryptic phosphopeptides depended on pH similarly. The pH dependence suggested that phosphate was linked to the N3 of a histidine residue. Co-electrophoresis and co-chromatography of alkaline hydrolysates of the labeled phosphoenzyme prepared from either substrate allowed the definitive identification of 3-phosphohistidine in the degradation products. Modification of the enzyme with diethylpyrocarbonate inactivated both the phosphotransferase and phosphohydrolase activities and suppressed the phosphorylation of the enzyme by ATP and fructose-6-P or by fructose-2,6-P2. Trypsin digestion of the phosphoenzyme formed upon incubation with ATP or fructose-2,6-P2 yielded an identical phosphopeptide after high pressure liquid chromatography. All the above data are consistent with this enzyme catalyzing both phosphohydrolase and phosphotransferase reactions with the mediation of phosphohistidine in the reaction scheme(s).

Structural and motional changes in glyceraldehyde-3-phosphate dehydrogenase upon binding to the band-3 protein of the erythrocyte membrane examined with [15N,2H]maleimide spin label and electron paramagnetic resonance.

Binding of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDHase; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating EC 1.2.1.12], to the cytoplasmic segment of band-3 protein in the erythrocyte (RBC) membrane has been examined by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies. GAPDHase, which was isolated from rabbit muscle and labeled with the resolution-enhancing deuterated N-(15N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide spin label ([15N,2H]MSL), showed the same binding specificity for the transmembrane band-3 protein of human erythrocyte membranes as reported for unlabeled GAPDHase from human RBC. Experimental EPR lineshapes from soluble and membrane-bound enzymes were analyzed by direct stimulation of spectra and indicated a structural alteration of the bound GAPDHase in the vicinity of the spin label, which was attached covalently to the active-site cysteine-149 residue. A rigorous theoretical analysis of the ST-EPR spectra of soluble and membrane-bound enzyme is presented and utilized in conjunction with model system analysis to demonstrate that the motion of membrane-bound GAPDHase could be characterized by an effective isotropic rotational correlation time of 20 microseconds. This indicated that the GAPDHase--band-4 complex exhibits motional freedom relative to the membrane-spanning segment of the band-3 protein or the RBC. The double substituted spin label [15N,2H]MSL affords gains in sensitivity and resolution that permit studies of membrane-bound enzymes at physiological levels and quantitative simulations of the EPR and ST-EPR lineshapes with reasonable computation times.

15N- and 2H-substituted maleimide spin labels: improved sensitivity and resolution for biological EPR studies.

The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of (15)N for (14)N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL). The EPR and ST-EPR spectra of the deuterated analogue [(2)H]MSL and the (15)N-substituted and deuterated derivative [(15)N, (2)H]MSL were compared with those of the parent MSL. The [(15)N, (2)H]MSL showed the greatest gain in sensitivity and the most marked sharpening of spectral features. These improvements were due to (i) a reduction in the spectral linewidths resulting from the relatively weak hyperfine interactions of the unpaired electron with deuterium and (ii) spectral simplification due to a reduction in the number of nuclear manifolds from three to two in replacing (14)N with (15)N. In the freely tumbling state, the spectra of [(15)N, (2)H]MSL and [(2)H]MSL showed 10-fold and 5-fold increases, respectively, in signal heights compared to MSL. To study the slow tumbling frequencies characteristic of biological molecules, the MSL and its derivatives were covalently bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase [GAPDHaase; D-glyceraldehyde-3-phosphate:NAD(+) oxidoreductase (phosphorylating), EC 1.2.1.12] on cysteine-149 of the catalytic site. The EPR and ST-EPR spectra of [(15)N, (2)H]MSL and [(2)H]MSL adducts showed 3- and 1.5-fold gains in sensitivity, respectively. More important, there were striking increases in resolution, particularly for [(15)N, (2)H]MSL over MSL. These improvements were observed throughout the correlation time range from 0.1 musec to 1 msec. The EPR spectrum of [(15)N, (2)H]MSL-GAPDHase at X-band showed no overlap of the two nuclear manifolds; therefore, all the elements of the A and g tensors could be measured directly from the spectrum. The increase in sensitivity and resolution of the (15)N- and deuterium-substituted spin labels permitted quantitative simulation of the EPR and ST-EPR spectra of a labeled protein. Computation time was reduced 90% by (15)N substitution. Use of (15)N-substituted and deuterated spin probes substantially improved characterization of the motional properties of a protein.