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Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH‐dependent reduction of uracil and thymine to the corresponding 5,6‐dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5‐fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2× 111 kDa) determined at 1.9 Å resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe–4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe–S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5‐fluorouracil reveals the architecture of the substrate‐binding sites and residues responsible for recognition and binding of the drug.

IntroductionThe 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. (6), aortic stenosis (7), dilated cardiomyopathy in the Syrian hamster (8), uninephrectomy plus steroid treatment (9), or the spontaneously hypertensive rat (10). The purpose of the present work was, therefore, to define performance, oxygen consumption, and parameters of energy reserve, i.e., tissue contents of ATP and creatine phosphate (CP), creatine kinase (CK) activity and isoenzyme distribution, and phosphoryl transfer rates via CK (using 3P-magnetization transfer), in normal rat heart and in residual intact myocardium after MI. Using these measurements, we directly tested whether changes in energy metabolism can contribute to contractile dysfunction in post-MI heart. MethodsAnimals and experimental MI. Infarcts or sham operations were carried out in 12-wk-old Wistar rats, kept in a 12-h light-dark cycle. Left anterior descending coronary artery (LAD) ligation was performed by a previously described technique (1, 11). Briefly, a left thoracotomy was performed under ether anesthesia and positive pressure ventilation. The heart was rapidly exteriorized by applying gentle pressure on both sides of the thorax. The LAD was ligated between the pulmonary outflow tract and the left atrium. The heart was then replaced into the thorax, lungs were inflated by increasing positive end-expiratory pressure, and the wound was closed immediately. Sham operation was performed using an identical procedure except that the suture was passed under the coronary artery without ligation. Mortality rate of infarcted rats for the first 24 h after the operation was 40-50%. Surviving rats were kept on commercial rat chow and water ad libitum. All procedures conformed to the guiding principles of the American Physiological Society. Isolated rat heart preparation. 8 wk after LAD ligation or sham operation, rats were anesthetized by injecting 20 mg pentobarbital sodium intraperitoneally. After thoracotomy, the heart was rapidly excised and immersed in ice-cold buffer. The aorta was dissected free and mounted onto a cannula attached to a perfusion apparatus, as described previously (12). Retrograde perfusion of the heart was started in the 1092Neubauer et al.J. Clin. Invest.C) The

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the final step in the biosynthesis of L-cysteine in Salmonella, viz., the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. UV-visible spectra of OASS exhibit absorbance maxima at 280 and 412 nm with pH-independent extinction coefficients over the range 5.5-10.8. Addition of OAS to enzyme results in a shift in the absorbance maximum from 412 to 470 nm, indicating the formation of an alpha-aminoacrylate Schiff base intermediate [Cook, P. F., & Wedding, R. T. (1976) J. Biol. Chem. 251, 2023]. The spectrum of the intermediate is also pH independent from 5.5 to 9.2. The observed changes in absorbance at 470 nm at different concentrations of OAS were used to calculate a Kd of 3 microM for OAS at pH 6.9. As the pH decreases, the Kd increases an order of magnitude per pH unit. The 31P NMR signal of the bound PLP has a pH-independent chemical shift of 5.2 ppm in the presence and absence of OAS. These results indicate that the phosphate group is present as the dianion possibly salt-bridged to positively charged groups of the protein. In agreement with this, the resonance at 5.2 ppm has a line width of 20.5 Hz, suggesting that the cofactor is tightly bound to the protein. The sulfhydrylase was also shown to catalyze an OAS deacetylase activity in which OAS is degraded to pyruvate, ammonia, and acetate. The activity was detected by a time-dependent disappearance of the 470-nm absorbance reflecting the alpha-aminoacrylate intermediate. The rate of disappearance of the intermediate was measured at pH values from 7 to 9.5 using equal concentrations of OAS and OASS. The rate constant for disappearance of the intermediate decreases below a pK of 8.1 +/- 0.1, reflecting the deprotonation of the active-site lysine that originally formed the Schiff base with PLP in free enzyme. A possible mechanism for the deacetylase activity is presented where the lysine displaces alpha-aminoacrylate which decomposes to pyruvate and ammonia.

The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda max of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L-serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda max of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L-serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L-cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine. The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen-bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The 31P NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)