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A Mozzarelli

Publications and source records attributed to A Mozzarelli.

51 records · Page 3Linked to original sources
PubMed1992

Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs.

The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine. The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue. In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme. These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure. As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit. Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed.

Amino Acid Sequence↗
PubMed1991

Crystals of haemoglobin with the T quaternary structure bind oxygen noncooperatively with no Bohr effect.

The relationship between the structure and function of haemoglobin has mainly been studied by comparing its X-ray crystal structures with its function in solutions. To make a direct comparison we have studied the functional properties of haemoglobin in single crystals, an approach that has been an important part of the investigation of several enzyme mechanisms. Here we report on the oxygen binding by single crystals of human haemoglobin grown in solutions of polyethylene glycol. Unlike haemoglobin crystals formed in concentrated salt solution, which crack and become disordered on oxygenation, crystals grown in polyethylene glycol remain intact. X-ray studies have shown that the T (deoxy) quaternary structure of haemoglobin in this crystal at pH 7.0 is maintained at atmospheric oxygen pressure, and that the salt-bridges are not broken. We find striking differences between oxygen binding by haemoglobin in this crystal and by haemoglobin in solution. Not only is oxygenation of the crystal noncooperative, but the oxygen affinity is independent of pH in the range 6.0-8.5, and is much lower than that of the T state in solution. The lack of cooperativity without a change in quaternary structure is predicted by the two-state allosteric model of Monod, Wyman and Changeux. The absence of a Bohr effect without breakage of salt-bridges is predicted by Perutz's stereochemical mechanism. In contrast to the X-ray result that oxygen binds only to the alpha haems, our measurements show that the alpha haems have only a slightly higher affinity than the beta haems.

Allosteric Regulation↗
PubMed1989

Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates.

Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.

Crystallization↗
PubMed1989

Coupled enzymatic reactions measured in a single protein crystal from myogen A.

Pairwise coupled reactions of fructose-1,6-bisphosphate aldolase and sn-glycerol-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and D-glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and sn-glycerol-3-phosphate dehydrogenase have been detected by microspectrophotometry in single crystals obtained from myogen A in the presence of polyethylene glycol. Microspectrophotometric measurements with polarized light demonstrate that the protein molecules are oriented and that NADH is bound with a definite orientation to the dehydrogenases within the crystal.

Animals↗
PubMed1987

Delay time of hemoglobin S polymerization prevents most cells from sickling in vivo.

A laser photolysis technique has been developed to assess the quantitative significance of the delay time of hemoglobin S gelation to the pathophysiology of sickle cell disease. Changes in the saturation of hemoglobin S with carbon monoxide produced by varying the intensity of a photolytic laser beam were used to simulate changes in the saturation of oxyhemoglobin S produced by variations in oxygen pressure. The presence of polymer at steady-state saturation with carbon monoxide was determined by measurement of the kinetics of gelation after complete photodissociation. The kinetics are a very sensitive probe for polymer since small amounts of polymerized hemoglobin increase the rate of nucleation sufficiently to eliminate the delay period. First, the equilibrium gelation properties of partially photodissociated carbonmonoxyhemoglobin S were shown to be the same as partially oxygenated hemoglobin S, and the method was then used to determine the effect of saturation on the formation and disappearance of polymers in individual sickle cells. The saturation at which polymers first formed upon deoxygenation was much lower than the saturation at which polymers disappeared upon reoxygenation. The results indicate that at venous saturations with oxygen, gelation takes place in most cells at equilibrium, but is prevented from occurring in vivo because the delay times are sufficiently long that most cells return to the lungs and are reoxygenated before polymerization has begun.

Anemia, Sickle Cell↗
PubMed1983

Interaction of a coenzyme analog with aspartate aminotransferase isoenzymes in the crystal.

The interaction between the coenzyme derivative 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate with cytoplasmic and mitochondrial apo-aspartate aminotransferase in the crystalline state was investigated to establish whether the structural differences, known to exist between the active sites of the two isoenzymes in solution, are maintained in the crystal although they are not apparent from the available crystallographic data. In the crystal, as in solution, both apo-isoenzymes reversibly bind the coenzyme derivative and catalyze a slow cleavage reaction, by which pyridoxal 5'-phosphate is produced and bound to the active-site lysine. In the case of the cytoplasmic isoenzyme, however, in the crystal as in solution, the initial complex can follow an alternative reaction path that leads to the formation of a covalent bond between the active-site lysine and the C-5 of the 2,4-dinitrophenyl moiety of the reagent. Therefore, crystal-packing forces neither abolish the active site properties that are needed to cleave the specifically bound reagent and are common to the two isoenzymes nor mask the subtle differences that allow for the selective irreversible labeling of the cytoplasmic isoenzyme.

Animals↗
PubMed1983

Complex formation and intermolecular electron transfer between flavocytochrome b2 in the crystal and cytochrome c.

The present study addresses the question whether tetrameric flavocytochrome b2 in the crystal is catalytically competent and, if so, whether it is possible to prepare a functional complex of the crystalline enzyme with its physiological electron acceptor cytochrome c. By single crystal microspectrophotometry we show that the native reduced enzyme can be oxidized by oxygen or ferricyanide and that the oxidized enzyme can be reduced by the electron donor L-lactate. Reduced cytochrome c appears to diffuse through the liquid channels of flavocytochrome b2 crystals and, at low ionic strength, to accumulate in amounts stoichiometrically equivalent to the enzyme protomers. Both cytochromes can be oxidized by ferricyanide. In the presence of L-lactate, both cytochromes become reduced. Since reduction of cytochrome c by L-lactate requires the catalytic action of flavocytochrome b2, it is concluded that the structure of the crystalline enzyme not only allows for electron transfer from L-lactate to flavin and intramolecular electron transfer from flavin to heme, but also for the formation of a productive complex with cytochrome c.

Crystallization↗
PubMed1979

Crystallographic symmetry and coenzyme binding properties of D-glyceraldehyde-3-phosphate dehydrogenase from the tail muscle of Palinurus vulgaris.

Crystals of apo- and holo-D-glyceraldehyde-3-phosphate dehydrogenase from the tail muscle of the Mediterranean lobster Palinurus vulgaris, previously found to be suitable for single crystal microspectrophotometric studies of catalytic activity in the crystalline state, have been examined by x-ray crystallography. The two forms are isomorphous, space group C 2 with cell dimensions a=128.4 A, b=99.9 A, c=80.3 A,beta=113.4 degrees. These data are consistent with a molecular weight of 73,000 in the crystallographic asymmetric unit, indicating that the tetrameric molecule possesses an exact 2-fold axis both in the presence and in the absence of NAD+. Analysis of the intensity distribution of conventional x-ray precession photographs shows that two further noncrystallographic diads are present and that the molecule has the 2 pseudo 22 symmetry found in other D-glyceraldehyde-3-phosphate dehydrogenases. Binding of NAD+ to apoenzyme in solution, at 25 degrees C, is anticooperative and it can be satisfactorily described by assuming two classes of coenzyme binding sites.

Animals↗
PubMed1979

Catalytic activity of aspartate aminotransferase in the crystal. Equilibrium and kinetic analysis.

Natural substrates and analogs rapidly diffuse through crystals of pig heart mitochondrial aspartate aminotransferase and react at the active sites causing spectral changes that can be measured by single-crystal microspectrophotometry. Dissociation constants for natural substrates and rate constants of transamination for slowly reacting substrates have been determined. A comparison between the data obtained in the crystal and in solution shows that the crystalline enzyme is catalytically competent and that events occurring in the crystal essentially parallel those occurring in solution, even though minor differences have been detected.

Animals↗