Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction.

The purpose of this study was to test the hypothesis that energy metabolism is impaired in residual intact myocardium of chronically infarcted rat heart, contributing to contractile dysfunction. Myocardial infarction (MI) was induced in rats by coronary artery ligation. Hearts were isolated 8 wk later and buffer-perfused isovolumically. MI hearts showed reduced left ventricular developed pressure, but oxygen consumption was unchanged. High-energy phosphate contents were measured chemically and by 31P-NMR spectroscopy. In residual intact left ventricular tissue, ATP was unchanged after MI, while creatine phosphate was reduced by 31%. Total creatine kinase (CK) activity was reduced by 17%, the fetal CK isoenzymes BB and MB increased, while the "adult" mitochondrial CK isoenzyme activity decreased by 44%. Total creatine content decreased by 35%. Phosphoryl exchange between ATP and creatine phosphate, measured by 31P-NMR magnetization transfer, fell by 50% in MI hearts. Thus, energy reserve is substantially impaired in residual intact myocardium of chronically infarcted rats. Because phosphoryl exchange was still five times higher than ATP synthesis rates calculated from oxygen consumption, phosphoryl transfer via CK may not limit baseline contractile performance 2 mo after MI. In contrast, when MI hearts were subjected to acute stress (hypoxia), mechanical recovery during reoxygenation was impaired, suggesting that reduced energy reserve contributes to increased susceptibility of MI hearts to acute metabolic stress.

Catabolism of pyrimidines in yeast: a tool to understand degradation of anticancer drugs.

The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides/antibiotics. During the last decade we have developed a yeast species, Saccharomyces kluyveri, as a model and tool to study the genes and enzymes of the pyrimidine catabolic pathway. In this report, we studied degradation of uracil and its putative degradation products in 38 yeasts and showed that this pathway was present in the ancient yeasts but was lost approximately 100 million years ago in the S. cerevisiae lineage.

Phosphorus-31 nuclear magnetic resonance study on cytoplasmic aspartate aminotransferase from pig heart. A reinvestigation.

Striking differences of the environment of the phosphate group of pyridoxal-P in cytoplasmic and mitochondrial aspartate aminotransferase have been reported. Since most details of the three-dimensional structure of the active sites of these isozymes are identical, it seemed difficult to rationalize the reported differences. Therefore, the cytoplasmic isozyme was reinvestigated using 31P-NMR at 72.86 MHz. The 31P chemical shift of the cofactor of this isozyme was found to be pH-dependent with a pKa of 6.2. In the presence of 100 mM succinate or 100 mM glutarate, the 31P chemical shift of bound pyridoxal-P remains at 4.71 or 4.79 ppm, respectively, in the pH range from 5.0 to 8.0, indicating that the phosphate group of the cofactor appears to be in its dianionic form. Reduction of the internal pyridoxal-P Schiff's base dramatically increases the pKa of the phosphate group of the phosphopyridoxyl moiety of the protein to 8.3. Hence, our results on the cytoplasmic isozyme are similar to those reported for the mitochondrial isozyme.

Temperature-dependent structural rearrangement of apotryptophanase in potassium phosphate.

Apotryptophanase (L-tryptophan indole-lyase, EC 4.1.99.1) from Escherichia coli B/1t7A shows, in the presence of potassium phosphate, a temperature-dependent structural rearrangement which is not observed in the presence of sodium phosphate or imidazole plus KC1. This rearrangement can be described by a two-state equilibrium between two forms of the apoenzyme. The midpoint temperature of the rearrangement (TM) and the van't Hoff enthalpy (delta H) at different potassium phosphate concentrations and pH values, respectively, were determined by measuring the temperature-dependence of the ultraviolet absorbance of apotryptophanase. Increasing the potassium phosphate concentration at pH 7.8 causes a simultaneous increase in total absorbance and the delta H value, whereas the TM increases between pH 7.0 and 7.8 but starts to decrease at pH values above 7.8. In 0.1 M potassium phosphate at the pH optimum of the enzyme (7.8) TM and delta H were found to be 293.1 K and 167 kJ X mol-1, respectively. Moreover, the tyrosine residues of apotryptophanase dissociate in potassium phosphate and in imidazole plus KCl with pK values of 8.6 and 9.8, respectively, indicating that potassium phosphate favors the formation of tyrosinate. The rearrangement might be interpreted as the formation of specific hydrogen bonds between tyrosine and potassium phosphate which are ruptured at higher temperature. Such hydrogen bonds cannot be formed at all or only to a small extent in the presence of imidazole plus KCl or sodium phosphate. Those hydrogen bonds stabilize the structure of apotryptophanase. In contrast, holotryptophanase requires only K+ for enzymatic activity.

A study of binary complexes of Escherichia coli maltodextrin phosphorylase: alpha-D-glucose 1-methylenephosphonate as a probe of pyridoxal 5'-phosphate-substrate interactions.

The glucose 1-phosphate (Glc-1-P) analog alpha-D-glucose 1-methylenephosphonate (Glc-1-MeP) inhibits competitively Escherichia coli maltodextrin phosphorylases against Glc-1-P (Ki = 0.20 mM) but also Pi (Ki = 0.36 mM). Exchange of the active site residue Glu637 to Asp by site-directed mutagenesis abolishes inhibition only in the synthesis direction (S-mode), while the degradative direction (P-mode) was not affected. Structural and conformational differences of the S-mode versus P-mode were also revealed by 31P-NMR spectroscopy by comparing chemical shifts of the cofactor pyridoxal-P in binary complexes formed either in the presence of Glc-1-MeP or of arsenate. In contrast the apparent pK of pyridoxal-P in both binary complexes was closely similar. Again, the total chemical shift of pyridoxal-P in the synthesis mode respectively degradative mode was differently affected in the binary complexes of the Glu637Asp mutant enzyme. This supports the contention that differential binding of the substrates in the synthesis or the degradative mode changes the arrangement and mutual interactions of cofactor phosphate and substrate phosphates.

Changes in the Bohr effect due to pyridoxylation of the alpha-chain terminal amino groups of hemoglobin.

Deoxyhemoglobins substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at the N-terminal amino groups of the alpha-chains were investigated by 31P-NMR spectroscopy. Titration curves of the 5'-side-chains show a substantial increase in acid strength in alpha-pyridoxylated deoxyhemoglobins when compared to the corresponding CO-liganded hemoglobins. These derivatives therefore contain a new oxygenation-linked acid group which opposes the normal Bohr effect. The loss in stabilization of the monoanion of the phosphate or phosphonate group derived from the three-dimensional structure can account for the lower pK of this ionization in deoxyhemoglobin as compared to CO-liganded hemoglobin. The reduction in the Bohr effect caused by modification of the alpha-chains with pyridoxal 5'-deoxymethyl-enephosphonate is quantitatively equal to the expected contribution of alpha-chain N-terminal amino groups.

PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway.

Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source. Previously, we screened several yeasts for their ability to catabolise pyrimidines. One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines. Here, a series of molecular techniques have been modified to clone pyrimidine catabolic genes, study their expression and purify the corresponding enzymes from this yeast. The pyd2-1 mutant, which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S. kluyveri genomic library. The complementing plasmid contained the full sequence of the PYD2 gene, which exhibited a high level of homology with mammalian DHPases and bacterial hydantoinases. The organisation of PYD2 showed a couple of specific features. The 542-codons open reading frame was interrupted by a 63 bp intron, which does not contain the Saccharomyces cerevisiae branch-point sequence, and the transcripts contained a long 5' untranslated leader with five or six AUG codons. The derived amino acid sequence showed similarities with dihydroorotases, allantoinases and uricases from various organisms. Surprisingly, the URA4 gene from S. cerevisiae, which encodes dihydroorotase, shows greater similarity to PYD2 and other catabolic enzymes than to dihydroorotases from several other non-fungal organisms. The S. kluyveri DHPase was purified to homogeneity and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product. The enzyme is a tetramer, likely consisting of similar if not identical subunits each with a molecular mass of 59 kDa. The S. kluyveri DHPase was capable of catalysing both dihydrouracil and dihydrothymine degradation, presumably by the same reaction mechanism as that described for mammalian DHPase. On the other hand, the regulation of the yeast PYD2 gene and DHPase seem to be different from that in other organisms. DHPase activity and Northern analysis demonstrated that PYD2 expression is inducible by dihydrouracil, though not by uracil. Apparently, dihydrouracil and DHPase represent an important regulatory checkpoint of the pyrimidine catabolic pathway in S. kluyveri.

Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli.

Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion. The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A. The asymmetric unit cell contains one protein molecule with Mr = 48,289. The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.

Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium.

The last step in cysteine biosynthesis in enteric bacteria is catalyzed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase. Here we report the crystal structure at 2.2 A resolution of the A-isozyme of O-acetylserine sulfhydrylase isolated from Salmonella typhimurium. O-acetylserine sulfhydrylase shares the same fold with tryptophan synthase-beta from Salmonella typhimurium but the sequence identity level is below 20%. There are some major structural differences: the loops providing the interface to the alpha-subunit in tryptophan synthase-beta and two surface helices of tryptophan synthase-beta are missing in O-acetylserine sulfhydrylase. The hydrophobic channel for indole transport from the alpha to the beta active site of tryptophan synthase-beta is, not unexpectedly, also absent in O-acetylserine sulfhydrylase. The dimer interface, on the other hand, is more or less conserved in the two enzymes. The active site cleft of O-acetylserine sulfhydrylase is wider and therefore more exposed to the solvent. A possible binding site for the substrate O-acetylserine is discussed.

Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy.

The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered [(1990) FEBS Lett. 266, 17-20; (1992) Biochemistry (in press)]. In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy. The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity. The results provide further evidence that thiamine pyrophosphate is bound primarily via the beta-phosphate moiety. In addition, a phosphoserine has been discovered in two of the four subunits.

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the beta-adrenoceptor.

The C-terminal part of the third intracellular loop of the beta-adrenoceptor is capable of stimulating adenylate cyclase in the presence of phospholipid vesicles via the stimulatory guanine nucleotide binding protein (Gs) [Palm et al. (1989) FEBS Lett. 254, 89-93]. We have investigated the structure of synthetic peptides corresponding to residues 284-295 of the turkey erythrocyte adrenoceptor in micelles, trifluoroethanol and aqueous solution, by using 2D 1H NMR and CD. In the presence of phospholipid micelles the peptides display a C-terminal alpha-helical region, whereas the N-terminal part was found to be highly flexible.

Rotational dynamics of 4-aminobutyrate aminotransferase.

The fluorescence dye 1-anilinonaphthalene-8-sulfonate (ANS) was used as a probe of non-polar binding sites in 4-aminobutyrate aminotransferase. ANS binds to a single binding site of the dimeric protein with a Kd of 6 microM. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (phi = 65 ns) corresponds to the rotation of a rather rigid dimeric structure. The microenvironment surrounding the natural probe pyridoxal-5-P covalently bound to the dimeric structure was explored using 31P-NMR at 72.86 MHz. In the native enzyme, the pyridoxal-5-P 31P-chemical shift is pH-independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the 31P-spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (phi = 40 ns). It is concluded that the phosphate of the pyridoxal-5-P molecule is rigidly bound to the active site of 4-aminobutyrate aminotransferase.

A preparative method for the isolation of genetic variant forms of glucosephosphate isomerase and a study of five variants.

A method has been developed for the rapid, quantitative separation of normal and abnormal glucosephosphate isomerase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Five different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. While the physical and catalytic properties of most of the variants were similar, clear differences in the stability of the allozymes were apparent. In order to detect mutations affecting the stability, a series of different stability tests are required.

NMR studies and semi-empirical energy calculations for cyclic ADP-ribose.

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.

A new expression vector for production of enzymes in the yeast Saccharomyces (Lachancea) kluyveri.

We overexpressed and purified enzymes involved in the pyrimidine catabolic pathway in the yeast Saccharomyces (Lachancea) kluyveri. A new vector was therefore designed, providing the first specific expression system in Saccharomyces kluyveri. The URC1 gene was overexpressed and a soluble protein obtained and successfully purified using the C-terminally added His-tag. Our system will be used for further studies of the structure and function of the enzymes belonging to the URC pyrimidine degradation pathway.

Degradation of pyrimidines in Saccharomyces kluyveri: transamination of beta-alanine.

Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have identified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding enzyme beta -alanine aminotransferase ((Sk)Pyd4p), highly homologous to eukaryotic gamma-aminobutyrate aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase ((Sk)Uga1p) with 80% and (Sk)Pyd4p with 55% identity to S. cerevisiae GABA-AT. (Sk)Pyd4p is a typical pyridoxal phosphate-dependent aminotransferase, specific for alpha-ketoglutarate (alpha KG), beta-alanine (BAL) and gamma-aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving two half-reactions and substrate inhibition. (Sk)Uga1p accepts only alpha KG and GABA but not BAL, thus only (Sk)Pydy4p belongs to the uracil degradative pathway.

Ischemic tolerance of human skeletal muscle.

Until now, the ischemic tolerance of muscle tissue has not been adequately understood. Even when muscle vitality is lost, the perfusion matrix of the muscle flaps is retained. Because of toxic decomposition, however, irreversibly damaged muscle cells almost certainly increase the rate of complications. The retention of the vitality of the transplanted muscle tissue is absolutely essential for the myokinetic substitute operations, currently in the development stage, involving the free transplantation of muscles. Investigations into vitality reserves were carried out on skeletal muscle specimens. Nuclear magnetic resonance spectroscopy was used to establish that, in ischemia, the ATP pool remained topped up to a large extent as long as phosphocreatine was available. As long as the ATP pool was retained, rearterialization led to the complete restoration of the essential preischemic metabolite concentrations. After the ATP had been exhausted, biochemical restitution through arterial reperfusion did not occur. The time by which the established vitality threshold was reached because of the loss of the ATP pool is called the critical ischemia time; it depends on muscle temperature. The critical ischemia time of human skeletal muscles was determined between 26 degrees and 38 degrees C. A normothermia of 34 degrees C yielded a critical ischemia time of 2.25 hours, which is shorter than that previously reported in the literature. An ischemic tolerance of 5 hours presupposes a muscle temperature of less than 26 degrees C.

Plasticity of the tryptophan synthase active site probed by 31P NMR spectroscopy.

The functional properties of tryptophan synthase alpha2beta2 complex are modulated by a variety of allosteric effectors, including pH, monovalent cations, and alpha-subunit ligands. The dynamic properties of the beta-active site were probed by 31P NMR spectroscopy of the enzyme-bound coenzyme pyridoxal 5'-phosphate. The 31P NMR signal of the cofactor phosphate of the internal aldimine exhibits a single peak at 3.73 ppm with a line width of 12 Hz. In the presence of saturating concentrations of sodium ions, the 31P signal shifts to 3.97 ppm concomitant with a change in line width to 35 Hz. The latter indicates that sodium ions decrease the conformational flexibility of the coenzyme. In the absence of ions, lowering pH leads to the appearance of a second peak at 4.11 ppm, the intensity of which decreases in the presence of cesium ions. Addition of L-serine in the presence of sodium ions leads to the formation of the external aldimine, the first metastable catalytic intermediate. The 31P signal does not change its position, but a change in line width from 35 to 5 Hz is observed, revealing that this species is characterized by a considerable degree of rotational freedom around the coenzyme C-O bond. In the presence of L-serine and either cesium ions or the allosteric effector indole-3-acetylglycine, the accumulation of the second catalytic intermediate, alpha-aminoacrylate, is observed. The 31P signal is centered at 3.73 ppm with a line width of 5 Hz, indicating that the phosphate group of the coenzyme in the external aldimine and the alpha-aminoacrylate exhibits the same flexibility but a slightly different state of ionization. Because the alpha-aminoacrylate intermediate but not the external aldimine triggers the allosteric signal to the alpha-subunit, other portions of the beta-active site modify their dynamic properties in response to the progress of the catalytic process. A narrow line width was also observed for the quinonoid species formed by nucleophilic attack of indoline to the alpha-aminoacrylate. The 31P signal moves downfield to 4.2 ppm, indicating a possible change of the ionization state of the phosphate group. Thus, the modification of either the ionization state of the coenzyme phosphate or its flexibility or both are, at least in part, responsible for the conformational events that accompany the catalytic process.

Resolution of pyridoxal 5'-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site.

A procedure has been developed to prepare the apoenzyme of O-acetylserine sulfhydrylase (apoOASS) by first converting the native enzyme to the alpha-aminoacrylate intermediate and dialyzing against 5 M guanidinium chloride. Aposulfhydrylase is stable for at least a month in buffers containing phosphate or phosphate analogues. Reconstitution of aposulfhydrylase with pyridoxal 5'-phosphate (PLP), 2'-methyl PLP (2'-MePLP), and pyridoxal 5'-deoxymethylenephosphonate (PDMP) results in enzymatically competent proteins. Pyridoxal in the absence and presence of phosphate and pyridoxal 5'-phosphate monomethyl ester are unable to form a Schiff base with apoOASS. The reconstitution of apoOASS with PLP is highly cooperative judged by the initial rate of activity regained and shows no evidence of saturation with PLP. The reconstituted enzymes have been studied using 31P NMR spectroscopy. The 31P NMR of the aposulhydrylase reconstituted with PLP exhibits a chemical shift of 5.2 ppm, identical to that of native enzyme. The latter has been interpreted in terms of a strong ionic interaction between enzyme and the 5'-phosphate of PLP (P. F. Cook, S. Hara, S. Nalabolu, and K. D. Schnackerz, 1992, Biochemistry 31, 2298-2303). Reconstitution with 2'-MePLP gives a lower chemical shift of 4.95 ppm, suggesting a weaker ionic interaction at the 5'-phosphate when compared to native enzyme. The PDMP-reconstituted enzyme gives a chemical shift of 23.7 ppm, consistent with the monoanionic form of the bound phosphonate. All of the chemical shifts are pH independent. The apoenzyme has also been reconstituted with pyridoxal 5'-sulfate. Although the resulting enzyme is not active in the overall reaction, it forms the external Schiff base. The PDMP- and 2'-MePLP-reconstituted enzymes have also been studied in the presence of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS.