Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase.

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.

Dissociation of mitochondrial malate dehydrogenase into active soluble subunits.

Gel exclusion chromatographic studies demonstrate that cytosolic and mitochondrial malate dehydrogenases (cMDH and mMDH) dissociate into subunits in the presence of 0.1% of the non-ionic detergent Triton X-100 (TX-100). The presence of cofactor and catalytically competent cofactor-substrate pairs does not protect mMDH against this dissociation. In contrast, cMDH dimers resist dissociation in the presence of either addition. Since steady state kinetic studies indicate both enzymes are fully active in the presence of 0.1% TX-100, we conclude that quaternary structure is not a kinetically important feature of mMDH structure and cooperativity does not account for mMDH kinetic anomalies. In contrast, cooperativity is a reasonable explanation for cMDH kinetic properties even in the presence of 0.1% TX-100, since this enzyme's subunits associate in the presence of active site ligands. The existence of fully active mMDH subunits raises the possibility that this species rather than the dimer may be a constituent of proposed multi-enzyme complexes of the mitochondrion. Preliminary chromatographic experiments involving gently disrupted mitochondria have found MDH activity in differently sized complexes, all with molecular weights larger than the mMDH dimer but smaller than complexes anticipated for multi-enzyme complexes involving other enzymes and the mMDH dimer.

The secondary structure of a fourteen-nucleotide fragment of the hairpin ribozyme.

A fourteen nucleotide RNA has been synthesized and its secondary structure investigated by non-denaturing polyacrylamide gel electrophoresis and 1H nuclear magnetic resonance spectroscopy. This fourteen nucleotide RNA corresponds to the hairpin loop end of the proposed secondary structure of the (-)sTRSV hairpin ribozyme. Non-denaturing polyacrylamide gel electrophoresis indicates that the fourteen nucleotide RNA exists predominantly as a monomer at a 1 mM strand concentration. Four peaks are found in the imino hydrogen region of the 1H NMR spectrum of the fourteen nucleotide RNA at this concentration. One-dimensional nuclear Overhauser effect spectroscopy of the imino hydrogen region of the 1H NMR spectrum gives results consistent with a model of the secondary structure of the fourteen nucleotide RNA having a three nucleotide hairpin loop and two double-stranded stems separated by a single bulged adenosine.

Cooperativity in the mechanism of malate dehydrogenase.

Cooperativity in the catalytic mechanism of porcine cytoplasmic malate dehydrogenase (sMDH) has been a point of ongoing discussion. Though previous investigations revealed little evidence of cooperativity, chemical modification studies reported by this laboratory demonstrate that binding of cofactor or cofactor plus substrate causes the enzyme's subunits to become chemically nonidentical. Therefore, we have reexamined the enzyme's steady-state kinetic and ligand-binding properties. To aide in characterizing sMDH kinetics, activities of the native enzyme and of sMDH, which was partially inactivated by an active-site-specific reagent, were examined. As expected for a negatively cooperative enzyme, steady-state kinetics (at pH 8.0, the pH optimum of the enzyme) are characterized by concave Eadie-Hofstee plots. Further, qualitative as well as quantitative results from partially inactivated sMDH strongly support negative cooperativity and eliminate many alternative mechanisms. Finally, results from equilibrium binding experiments are consistent with cytoplasmic malate dehydrogenase binding NADH in a negatively cooperative manner. Together, these results indicate that cytoplasmic malate dehydrogenase acts as a negatively cooperative enzyme.

Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1.

Rhizobium strain BTAi 1, which nodulates both stems and roots of Aeschynomene indica L., formed bacteriochlorophyll and photosynthetic reaction centers resembling those of purple photosynthetic bacteria when grown aerobically ex planta under a light-dark cycle. Bacteriochlorophyll formation was not observed under continuous dark or light growth conditions. The amount of pigment formed was similar to that previously found in aerobic photosynthetic bacteria. Stem nodules appear to fix nitrogen photosynthetically, as illumination of A. indica stem nodules with near-infrared light resulted in an enhanced rate of acetylene reduction. Near-infrared light did not enhance acetylene reduction when either A. indica or soybean root nodules were illuminated. The BTAi 1 isolate can be differentiated from members of the family Rhodospirillaceae by several criteria.

Mutation of essential catalytic residues in pig citrate synthase.

Two amino acid residues, His274 and Asp375, were replaced singly in the active site of pig citrate synthase (PCS) with Gly274, Arg274, Gly375, Asn375, Glu375, and Gln375. The nonmutant protein and the mutant proteins were expressed in and purified from Escherichia coli, and the effects of these amino acid substitutions on the overall reaction rate and conformation of the PCS protein were studied by initial velocity and full time course kinetic analysis, behavior during affinity column chromatography, and monoclonal antibody reactivity. Native and mutant proteins purified similarly had a subunit molecular weight of 50,000 and were homologous when examined with 10 independent a-PCS monoclonal IgGs or with a polyclonal anti-PHCS serum. No activity was detected for Asn375 or Gln375. The kcats of the other purified mutant proteins, however, were decreased by about 10(3) compared to the nonmutant enzyme activity. The Km for oxalacetate was decreased 10-fold in the Glu375 protein and was reduced by half in Gly274 and Arg274 PCSs, while the Km for acetyl-CoA was decreased 2-3-fold in Gly274, Arg274, and Gln375 PCSs. A mechanism is proposed that electrostatically links His274 and Asp375.

Ligand-induced asymmetry between active sites of cytoplasmic malate dehydrogenase: a chemical modification study.

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.

Comparison of solid and solution state protein structures. Photoacoustic study of solid state bovine methemoglobin derivatives.

In an effort to examine the consequences of phase state changes on protein structures, optical absorption spectra of several bovine hemoglobin derivatives in lyophilized, ammonium sulfate-precipitated, and crystalline states were examined. Absorption spectra were used to compare protein conformations. Differences between solution and several solid state spectra were apparent. They were greatest for lyophilized preparations, were inhibited by ligand binding, the tightest binding ligand being the most effective inhibitor, and were substantially or totally reversible upon dissolving the solid state protein. Though the magnitude of spectral changes varied, they all indicated characteristically different proportions of the protein were converted to a reversible hemichrome in each type of solid state preparation. The extent of hemichrome formation was correlated with the degree to which the protein tends to be dehydrated in each solid state preparation. This may reflect the role of water in determining the three-dimensional structure of hemoglobin. Absorption spectra of solid state hemoglobin preparations were obtained from photoacoustic spectra using a novel method for spectral analysis. Results presented here demonstrate the utility of this procedure in probing structures of solid state proteins.

Comparison of solution and crystalline state protein structures. Photoacoustic study of horse and human hemoglobins.

In an effort to assess the influence that crystallization may have on protein conformations, optical absorption spectra of crystalline state hemoglobin derivatives have been examined. These spectra were obtained from photoacoustic spectra using a computer-assisted analysis. Comparisons of crystal and solution state hemoglobins using crystal minus solution state difference spectra indicate that the conformations of these proteins are similar in both states. Crystallization does not change the absorption properties of horse oxyhemoglobin or the cyanide and azide adducts of horse and human methemoglobin. Spectra of crystalline methemoglobins, which are prepared by ligand exchange in the crystalline state, are identical to the spectra of the final crystalline state adduct prepared without ligand exchange. Further, the allosteric effector, inositol hexaphosphate, causes the same spectral changes in solution and crystalline state hemoglobins. However, differences between crystal and solution state spectra of both fluoride and aquo methemoglobin are observed. These differences suggest that small but perhaps functionally significant changes in the heme regions of these derivatives accompany crystallization.

Crystallization-induced modification of cytoplasmic malate dehydrogenase structure and function.

In an effort to assess the effects of phase-state changes on protein conformations, we have compared several properties of cytoplasmic malate dehydrogenase in the crystalline and solution states. Two crystalline forms of the enzyme have been examined: one crystallized in the presence of nicotinamide adenine dinucleotide and the other in its absence. Though both forms catalyze cytoplasmic malate dehydrogenase's normal substrate conversions, they have specific activities 150-3000-fold less than the solution-state enzyme. These dramatic activity decreases cannot be accounted for by diffusion constraints imposed by the crystal lattice nor do they result from the manipulations necessary to crystallize or cross-link the enzyme. Further, crystal- and solution-state enzymes have different pH dependences of their enzymatic activities, have different sensitivities toward inactivation by the covalent inhibitor iodoacetate, and respond differently to nicotinamide adenine dinucleotide protection against this inactivation. Finally, crystals of the enzyme grown in the presence and absence of cofactor are also distinguishable from one another by using the same criteria. Taken together, these results suggest that crystallization perturbs the dynamics and, perhaps, the average conformation of cytoplasmic malate dehydrogenase.

Computer-assisted analysis of photoacoustic spectra of solid and solution state cyanide methemoglobin.

A computer-assisted method for analyzing photoacoustic spectra has been developed. Using this analysis, the relative absorption spectrum and either the chromophore concentration or thermal diffusivity characteristic of a sample can be derived from its photoacoustic spectrum. We have demonstrated the accuracy of the method by analyzing photoacoustic spectra of solution and crystalline-phase bovine cyanide methemoglobin. BASIC and FORTRAN routines used to collect and to analyze photoacoustic spectra are described. Photoacoustic spectroscopy can be used in conjunction with the analytical method presented here to obtain accurate absorption spectra from a variety of solid, opaque, and/or turbid samples.

Saccharide binding to transition metal ion free concanavalin A.

Saccharide binding has been observed with demetallized concanavalin A in the presence of Ca(2+) only, using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside. At pH 7.2 both the nicked and intact forms of concanavalin A bound 4-methylumbelliferyl alpha-D-mannopyranoside with similar affinities. Competitive binding with methyl alpha-D-mannopyranoside was demonstrated. The association constants at 5 degrees C were 9.6 +/- 0.6 X 10(4) M(-1) for 4-methylumbelliferyl alpha-D-mannopyranoside and 1.1 +/- 0.3 X 10(4) M(-1) for methyl alpha-D-mannopyranoside. 4-Methylumbelliferyl alpha-D-mannopyranoside binding was also observed if demetallized concanavalin A was remetallized with less than stoichiometric amounts of Ca(2+). The association constants with low Ca(2+) concentrations were similar to those determined with saturating Ca(2+). With less than stoichiometric levels of Ca(2+), the number of sugar molecules bound per protein subunit was a reflection of the fraction of activated lectin subunits. These results show that saccharide binding activity of concanavalin A does not require a transition metal ion at pH 7.2; only Ca(2+) is required. At pH values near 5, where most previous studies have been carried out, both a transition metal ion and Ca(2+) are necessary.

Calcium-induced cooperativity of manganese binding to concanavalin A.

Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.

Kinetic properties of carboxypeptidase B in solutions and crystals.

The correlation of the structure of crystalline enzymes with their activities in solution assumes that the catalytic properties are identical in the two physical states. The present data demonstrate that in bovine carboxypeptidase B they differ significantly. Normal Michaelis-Menten kinetics characterize the hydrolysis of several esters and peptide substrates in both physical states. Crystallization reduces kcat 16 to 320-fold, while it affects KM variably and less dramatically. Small molecules inhibit catalytic activity both in solution and in crystals, but the carboxypeptidase inhibitor from potatoes (molecular weight 4200) does no inhibit the crystals. The activities of bovine carboxypeptidases A and B toward identical substrates are more similar in their crystals than in their solutions. This suggests that, over and above the structural dissimilarity of their crystals, conformational differences may additionally determine the activities of the two enzymes in solution. The findings demonstrate that the catalytic properties of carboxypeptidase B depend critically on its physical state.