Cold inactivation and dissociation into dimers of Escherichia coli tryptophanase and its W330F mutant form.

The kinetics and mechanism of reversible cold inactivation of the tetrameric enzyme tryptophanase have been studied. Cold inactivation is shown to occur slowly in the presence of K+ ions and much faster in their absence. The W330F mutant tryptophanase undergoes rapid cold inactivation even in the presence of K+ ions. In all cases the inactivation is accompanied by a decrease of the coenzyme 420-nm CD and absorption peaks and a shift of the latter peak to shorter wavelengths. The spectral changes and the NaBH4 test indicate that cooling of tryptophanase leads to breaking of the internal aldimine bond and release of the coenzyme. HPLC analysis showed that the ensuing apoenzyme dissociates into dimers. The dissociation depends on the nature and concentration of anions in the buffer solution. It readily occurs at low protein concentrations in the presence of salting-in anions Cl-, NO3- and I-, whereas salting-out anions, especially HPO4(2-), hinder the dissociation. K+ ions do not influence the dissociation of the apoenzyme, but partially protect holotryptophanase from cold inactivation. Thus, the two processes, cold inactivation of tryptophanase and dissociation of its apoform into dimers exhibit different dependencies on K+ ions and anions.

Interactions of Escherichia coli tryptophanase with quasisubstrates and monovalent cations studied by the circular dichroism and fluorescence methods.

The reaction of tryptophanase and its W330F and W248F mutant forms with quasi-substrates forming an external pyridoxal phosphate aldimine or quinonoid is accompanied by the appearance of a positive circular dichroism (CD) peak at 290 nm. The peak seems to arise from a Tyr residue undergoing reorientation during the reaction. The peak does not appear upon formation of non-covalent Michaelis complexes of the enzyme with quasi-substrates such as indolepropionate, beta-phenyllactate and alpha-methylphenylalanine. The non-covalent complexes and external aldimines exhibit similar absorption spectra but can be distinguished by their CD and by the intensity of their fluorescence. Formation of the non-covalent complexes leads to an increase in positive CD at 420 nm while formation of the external aldimines leads to disappearance of the positive CD at 420 nm and its replacement by negative CD; it also leads to strong quenching of the coenzyme fluorescence at 500 nm. The quantum yield of fluorescence of the external aldimines is 6-times lower than that of the internal aldimine. Activating cations (K+, NH4+) strongly diminish the intensity of a negative protein CD band at 275 nm. From a comparison of the intensity of this band in the spectra of the wild-type holo- and apoenzyme and in the tryptophan mutants, it was deduced that the band belongs to a Tyr residue, which may be a part of the cation-binding site or located in its immediate vicinity.

Crystal structure of the closed form of chicken cytosolic aspartate aminotransferase at 1.9 A resolution.

The crystal structure of chicken cytosolic aspartate aminotransferase (cAATase; EC 2.6.1.1) has been solved and refined at 1.9 A resolution. Orthorhombic crystals, space group P2(1)2(1)2(1), a = 56.4 A, b = 126.0 A and c = 142.3 A, were grown from polyethylene glycol solutions in the presence of maleate, a dicarboxylic inhibitor that forms a Michaelis-like complex. The pyridoxal form of the enzyme was used for crystallization. Diffraction data were collected using synchrotron radiation. The structure of the new orthorhombic crystal form was solved by molecular replacement using the partially refined 2.8 A resolution structure of the high-salt crystal form as a search model. The final value of the crystallographic R-factor after rigid body and restrained least-squares refinement is 0.175 with very good model geometry. The two 2-fold-related subunits of cAATase have distinct environments in the crystal lattice. Domain movement is strictly hindered by the lattice contacts in one subunit, while the second one possesses conformational freedom. Despite their different environments, both subunits were found in the closed conformation with one maleate molecule tightly bound in each active site. The present study allows a detailed comparison of the highly refined structures of the aspartate aminotransferase isozymes, and thus provide better insight into the role of conserved and variable residues in substrate recognition and catalysis.

Linear dichroism studies of tryptophanase and its quasisubstrate complexes oriented in polyacrylamide gel.

Tryptophanase from Escherichia coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra have been measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305-nm band apparently belongs to an aromatic amino acid residue. The 490-nm band disappears after treatment with NaBH4 or after incubation with L-alanine and subsequent dialysis. It is suggested that the 490-nm band belongs to a quinonoid enzyme subform. The reaction of tryptophanase with threo-3-phenyl-DL-serine, L-threonine and D-alanine leads to formation of an external aldimine with an intense absorption band at 420-425 nm. The values of reduced LD (delta A/A) in this band strongly differ from that in the 420-nm band of the free enzyme. The LD value of the complex with D-alanine is intermediate between those of the free enzyme and the complex with 3-phenylserine. In the presence of indole the complex with D-alanine displays the same LD as that observed with 3-phenylserine. The reaction of tryptophanase with L-alanine or oxindolyl-L-alanine leads to formation of a quinonoid intermediate with an absorption band near 500 nm. The LD value in this band is close to that of an external aldimine with L-threonine. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.

Conformational changes in the active site of tryptophanase revealed by the circular dichroism method.

Tryptophanase from E. coli displays positive CD in the coenzyme absorption bands at 337 and 420 nm. Breaking of the internal coenzyme-lysine imine bond upon reaction with hydroxylamine or amino-oxyacetate is accompanied by a strong diminution of the positive CD. Interaction of tryptophanase with L-threonine and beta-phenyl-DL-serine(threo form) leads to a decrease in absorbance at 337 nm and to an increase at 425 nm. This is associated with inversion of the CD sign, i.e. with disappearance of the positive CD in the 420-nm band and its replacement by a negative CD. L-Phenylalanine, alpha-methyl-DL-serine and D-alanine cause an increase in absorbance at 425-430 nm and a diminution of the positive CD in this band. In the presence of D-alanine and indole a negative CD appears in the 400-450 nm region. It is inferred that an external coenzyme-quasisubstrate aldimine is formed on interaction of the above amino acids with the enzyme. L-Alanine and oxindolyl-L-alanine evoke an intense narrow absorption band at 500 nm ascribed to a quinonoid intermediate; a positive CD is observed in this band. The dissymmetry factor delta A/A in the 500-nm band is much smaller than that in the absorption bands of the unliganded enzyme. Inversion of the CD sign on formation of the external aldimine and diminution of the dissymmetry factor in the quinonoid band indicate that reorientations of the coenzyme occur in the course of the catalytic action of tryptophanase.

Coenzyme reorientations in the active sites of aspartate aminotransferase isoenzymes studied by linear dichroism method.

Cytosolic and mitochondrial pig heart aspartate aminotransferases (cAspAT and mAspAT) and chicken heart cAspAT have been oriented in a compressed slab of polyacrylamide gel and their linear dichroism LD spectra have been recorded. The coenzyme's tilt angles in the active sites of chicken cAspAT and pig mAspAT and their quasisubstrate complexes imitating catalytic intermediates have been computed. The computations are based on reduced linear dichroism values (delta A/A), the known directions of the transition dipole moments in the coenzyme ring and atomic coordinates of the coenzyme obtained by X-ray crystallography. It has been found that formation of the enzyme complex with glutarate and protonation of the internal pyridoxal-lysine aldimine induce reorientations of the coenzyme. As a result of protonation, the coenzyme ring tilts by 27 degrees in cAspAT and 13 degrees in mAspAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAspAT and 39 degrees in mAspAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAspAT and 53 degrees in mAspAT. It is inferred that the basic features of the active site dynamics are similar in the three AspAT's studied. The differences in the coenzyme tilt angles between cAspAT and mAspAT may be linked to catalytic and structural peculiarities of the isoenzymes.

Phosphorus-31 nuclear magnetic resonance of aspartate aminotransferase from chicken heart cytosol.

31P-nuclear magnetic resonance and absorption spectra of cytosolic chicken aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been recorded in the pH range from 5 to 8.5. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; the chemical shift change was 0.35 ppm. The pK value found by spectrophotometric titration of the enzyme proved to be about 6.0. The monoanion-dianion transition of the 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in the 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein-bound coenzyme is in a dianionic form throughout the investigated pH range; the pH-dependence of the 31P chemical shift may be due to a conformational change at the active site. In the presence of 100 mM succinate, 6 mM aminooxyacetate or 25 mM cycloserine, the 31P chemical shift is insensitive to pH variations.

Reorientations of coenzyme in aspartate transaminase studied on single crystals of the enzyme by polarized-light spectrophotometry.

Investigation of polarized-light absorption spectra of single crystals of cytosolic aspartate transaminase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from chicken heart has revealed that the coenzyme's absorption bands at 430 and 360 nm are polarized in opposite directions, both in crystals of the free enzyme and in its complex with a quasi-substrate, 2-methylaspartate. The opposite signs of polarization of the 430 and 360 nm bands of the free enzyme indicate different orientation of the pyridine ring of pyridoxal 5'-phosphate in the protonated and non-protonated forms of the "internal" coenzyme-lysine aldimine. These data suggest that reorientation of the coenzyme ring occurs mainly in the first step of the catalytic reaction, associated with proton transfer from the NH+3 group of amino acid substrate to the coenzyme-lysine aldimine. Absorption bands at 333 and 430 nm are seen in the spectra upon soaking the crystals in solutions containing aspartate, glutamate or cysteinesulfinate. Both bands are polarized in the same direction as is the 430 nm absorption bands of the protonated internal aldimine. Soaking the crystals in solutions containing 2-oxoglutarate, glutarate or maleate reverses the sign of polarization of the 430 nm band. High concentrations of acetate induce the same effect. Thus, binding of dicarboxylate or acetate anions in the active site of aspartate transaminase appears to result in partial or complete return of the coenzyme ring to a position similar to that of the non-protonated internal aldimine.