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A pH-rate method, based on following the time dependence of hydroxide release by monitoring the pH drift as a funetion of time, is developed and applied to the construction of a detailed log activity vs pH profile for yeast pyruvate decarboxylase. In the pH range of 5 to 7 at 30°C this method reproduces the results of conventional initial rate studies but, in addition, produces a hitherto unreported pH independent activity region between pH 5.4–5.8. At least four pKs can be discerned in the pH-activity profile of this enzyme, instead of the two previously reported.

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 m m 2,3-butanedione. Inorganic phosphate, ribose-1-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-a-vis the fraction of arginines modified suggested that one essential arginine residue was being modified.

The log kobs vs. pH profiles were determined in the intermediate acidity region for the glycosyl hydrolysis of guanosine and its 8-amino, 8-monomethylamino, 8-dimethylamino and 8-bromo derivatives. The decreased rate of the 8-amino and enhanced rate of the 8-bromo compound compared to guanosine support an A type mechanism: base protonation followed by glycosyl bond cleavage. All three 8-amino guanosines exhibited log kobs - pH profiles clearly showing that both mono and di-base protonated nucleosides undergo hydrolysis. The 700 fold rate acceleration of 8-N(CH3)-guanosine compared to 8-NHCH3-guanosine and the 110 fold rate acceleration of 8-N(CH3)2-adenosine compared to 8-NHCH3-adenosine could be unequivocally attributed to the fixed syn glycosyl conformation of both 8-dimethylamino compounds and relief of steric compression upon hydrolysis in these molecules. The lack of anomerization of all substrates during the course of the reaction supports an A rather than a Schiff-base mechanism.

/sup 13/C NMR and /sup 31/P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-(1-/sup 13/C)- or D-(6-/sup 13/C)glucose as substrates, the major metabolic products identified from /sup 13/C NMR spectra were (2-/sup 13/C)ethanol, (3-/sup 13/C)alanine, and C/sub 1/- and C/sub 6/-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C/sub 6/- than from C/sub 1/-labeled glucose; (ii) C/sub 1/-labeled glucose yielded 19% C/sub 6/-labeled trehalose, while C/sub 6/-labeled glucose yielded only 4% C/sub 1/-labeled trehalose; (iii) a substantial transfer of /sup 13/C from C/sub 2/-labeled glucose to the C/sub 2/-position of ethanol. /sup 31/P NMR spectra showed millimolar levels of intracellular inorganic phosphate (P/sub i/), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic P/sub i/ and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100more » nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase in the rate of ethanol production; (ii) a 38% increase in the rate of alanine production; (iii) a 27% increase in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO/sub 2/ and glycogen, as previously shown.« less

Formulas are given allowing the calculation of electronic molecular energy up to the third-order in a perturbation theory using fully localized bond orbitals with zero differential overlap between them. The method is applied to small molecules using the CNDO approximations of Pople and Segal.

Extended Huckel molecular orbital calculations are reported on cyclic AMP and its phosphonate analog. A parallel Lennard-Jones potential and molecular orbital study on the barrier to rotation of the adenine base around the sugar phosphate moiety indicates a rather low barrier for both molecules studied (6.6 kcal/mole and less than 6 kcal/mole for the cyclic phosphate and cyclic phosphonate, respectively) with most favorable syn and anti forms possessing similar energies. According to the calculations this low barrier may be the most distinguishable property when compared to the molecule's precursor ATP and enzymically derived product 5′-AMP.

The new Peirce-General Electric copolymer membrane oxygenator combined with a Sarns roller pump was used clinically for the first time on 24 children undergoing open-heart operations. The children ranged in age from 5 to 48 months (mean 27 months) and weighed between 3.6 and 16 kg. (mean 9 kg.). The priming volume averaged 120 ml. of blood for the 1-square-meter oxygenator and 240 ml. for the 2-square-meter oxygenator. The duration of perfusion varied from 36 to 180 minutes (mean 96 minutes), depending upon the complexity of the repair. During perfusion, flow rates averaged 127 ml. per kilogram of body weight with a pO 2 average of 350 mm. Hg (range 54 to 670). There was no significant change in acid-base balance during or immediately after the perfusion. Serum hemoglobin averaged 13.1 mg. per 100 ml. preoperatively, increased to 61.4 mg. per 100 ml. at the completion of the perfusion, and decreased to 18.4 mg. per 100 ml. by 24 hours postoperatively. Platelet count preoperatively averaged 228,000 and decreased to 119,000 at the conclusion of the perfusion; it increased to an average of 129,000 twenty-four hours after operation. There was no excessive bleeding postoperatively resulting from coagulation defects. The copolymer membrane has been shown in this group of patients to be an effective, reliable, and safe oxygenator for total cardiopulmonary bypass.

Localized SCF orbitals are obtained by using the self-energy criteria in the framework of semiempirical methods (Pariser-Parr, Pople-Segal). The delocalisation problem is discussed. These localized orbitals (both occupied and virtual) are used for the calculation of the second-order correlation energy. Qualitative and quantitative features are discussed. The use of localized orbitals allows a deep reduction of the significant terms in the correlation energy.

Extended Huckel calculations have been performed for the structure of C5-C6 dihydrouracil and thymine and for the uracil and thymine photodimers. The results indicate, in agreement with experiment, that the most stable conformation of the dihydropyrimidines should be the half-chair one. The preferred conformations of the dimers should correspond to planar bases forming an angle of 120° with the cyclobutane ring. The most stable dimers, per se, appear to be the trans-anti and the trans-syn ones in the case of uracil and the cis-anti and the trans-anti ones in the case of thymine. Other factors than inherent stability must play a decisive role in the production of the dimers under specific conditions. When a semi-empirical reduction is carried out upon their absolute values the dipole moments predicted for the different conformers of the thymine dimer agree very satisfactorily with experiment.

The extended Huckel theory has been applied to the study of the conformation of the nucleosides of the purine and pyrimidine bases of the nucleic acids. Although the evaluation of the total energy as a function of the rotation angle presents in all cases two minima, the calculations predict a preferred anti conformation for uridine, cytidine and adenosine and a preferred syn conformation for guanosine. These predictions appear to be in agreement with the available experimental data.

Further reduction of Doppler tracking data from Mariner 9 confirms our earlier conclusion that the gravity field of Mars is considerably rougher than the fields of either the earth or the moon. The largest positive gravity anomaly uncovered is in the Tharsis region which is also topographically high and geologically unusual. The value obtained for the inverse mass of Mars is in good agreement with prior determinations from Mariner fly by trajectories. The direction found for the rotational pole of Mars is in excellent agreement with Sinclair's recent value, determined from earth-based observations of Mars' satellites. Other important physical constants that have either been refined or confirmed by the Mariner 9 data include: (1) the dynamical flattening, (2) the maximum principal moment of inertia, and (3) the period of precession of Mars' pole.

Perturbation theory in conjunction with the use of fully localized bond orbitals provides a precise and fast method for calculating electronic energy of molecules. This is illustrated in the framework of semi-empirical thoeries and the physical interpretations allowed by the method are shown.

The total energy and the electronic properties of some syn purine nucleosides have been determined as a function of the rotation angle around the C–N glycosidic linkage. Invariably, the crystallographic coordinates provide a state lying at or very near the minimum on the potential energy curve according to the extended Huckel parametrization here employed. The ribose moiety always carries a net positive charge whereas the base is always negatively charged. Changes in dipole moment magnitudes for the overall structure as well as in net atomic charges are noted upon rotation around the glycosidic linkage indicating that potential functions for polynucleotides may be in error if such changes are ignored.

The dipole moments of all the molecules were calculated and a qualitative relationship was found found between the dipole moment component along the C-H bond and the chemical shift of the associated proton.

We have studied the solvent dielectric constant dependence of the ionic association process in 2,3,4-trimethylthiazolium iodide as a model for the interaction of the thiazolium ring of thiamine with a carboxylate or other negatively charged species in enzymic processes requiring thiamine pyrophosphate as a co-factor. The Kass value for the ion pair (as measured by u.v. spectroscopy) increases as the solvent dielectric constant decreases from 3.4 in pure water to 175 in pure ethanol. The ion pair is subject to electrostatic stabilization as manifested by a linear log Kassvs. (dielectric constant)–1 plot. This type of interaction would be stabilized in a hydrophobic environment, and may have a multiple role in enzymic processes requiring the thiamine pyrophosphate coenzyme.

An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom.

Carbon-13 kinetic isotope effects were determined for the decarboxylation of pyruvate by thiamin and for the decarboxylation of 2-(1-carboxy-1-hydroxyethyl)-3,4-dimethylthiazolium chloride (a covalent pyruvate-thiamin adduct) in water and in aqueous ethanol. The kinetic isotope effect for the decarboxylation step in the second system increased from 1.051 in H/sub 2/O to approximately 1.058 in 50% v/v ethanol. The isotope effect in the thiamin catalyzed model reaction increased from the 0.992 inverse effect observed in H/sub 2/O to 1.007 in 50% aqueous ethanol. The rate of decomposition of the covalent thiamin-pyruvate adduct to reactants relative to its rate of decarboxylation is greater in ethanol than in water. Further, the results suggest that the changes of the vibrational force constants in going from the ground state to the transition state are greater in aqueous ethanol than in water.

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.

Several aspects of the stereoelectronic requirements of substrates of human erythrocytic purine nucleoside phosphorylase (E.C. 2.4.2.1) were elucidated providing the following information: (a) the N1 position cannot have a nonhydrogen substituent; (b) the 5'-OH group must be present for catalytic activity to be exhibited but is not an essential functional group for inhibitory action to be observed; (c) on the C8 position groups larger than -NH2 or -Br cannot be accommodated; (d) the syn-glycosyl conformation (i.e., 8-bromoguanosine) is acceptable but may not be an absolute requirement for phosphorolysis; (e) among nucleic base inhibitors methylation at N3, N7, or N9 vastly decreases the inhibitory properties as does a nitrogen in lieu of C-H in the 8 position. The results clearly indicate that this enzyme differs in its stereoelectronic requirements from the Escherichia coli enzyme.

A series of phenyl substituted E-4-phenyl-2-keto-3-butenoic acid derivatives were synthesized (p-Cl, m-Cl, p-NO2, m-NO2, o-NO2, 3,4-Cl2, 2,6-Cl2, p-CH3O, p-(CH3)2N) and tested as potential irreversible inhibitors of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1). All those derivatives with electron withdrawing substituents were found to be time-dependent inactivators of the enzyme, unlike the p-CH3O- and p-(CH3)2N derivatives. Detailed kinetic studies with the m-nitro derivative (the most potent inhibitor) indicated that this compound formed reversible complexes with the enzyme at two sites (supposed regulatory and catalytic with Ki values of 0.026 and 0.13 mM, respectively) prior to irreversible inactivation of the enzyme. In addition, concurrently with the inactivation, addition of the m-NO2 derivative to the enzyme produced a new VIS absorbance with lambda max near 430 nm. This absorbance was attributed to the enzyme-bound enamine intermediate. The time course of formation and disappearance of the intermediate could be determined and provided detailed information about the mechanism of the enzyme.

Several aspects of the co-enzyme function of thiamin pyrophosphate and Mg(II) in the action of brewer’s yeast pyruvate decarboxylase were examined. All experiments to be reported were performed by first resolving then reconstituting holoenzyme. The principal findings are the following: (1) An aminopyrimidine derivative and the 2,4-dinitrophenylether of thiamin were inhibitory; (2) upon reconstitution of apoenzyme with either pyrophosphate or Mg(II) there is an enhancement in the UV absorption between 300–400 nm and this enhancement develops with at, of ca 2 min. There is a lagtime in the regaining of the activity as well and it is demonstrated that the formation of the enhanced absorption is a necessary but not a sufficient condition for regaining of the activity; (3) reconstitution with Eu(III) in place of Mg(II) affords ca 50% of the activity compared to Mg(II) and leads to a large enhancement in the fluorescence emission spectrum which enhancement can be subsequently quenched by the addition of thiamin pyrophosphate. The fluorescence data for the apoenzyme-Eu(III) binary complex indicate the existence of two types of binding sites with Kd’s of 200 μm and (6±2) μm respectively. A qualitative assessment of the quenching curve by thiamin pyrophosphate indicates that there is an apparent Kd of the apoenzyme-Eu(III) complex of ca 1 μm.

The very low field proton nuclear magnetic resonance (1H NMR) found in aqueous solutions of serine proteases and their zymogens is characteristic of the hydrogen bond between the imidazolium and aspartate groups of the catalytic triad: Ser-His-Asp [Robillard, G., & Shulman, R. G. (1972) J. Mol. Biol. 71, 507--511]. According to 1H correlation NMR spectroscopic studies performed in 80/20 (v/v) H2O/2H2O, no such resonance is found in native subtilisins (even at -2 degrees C and pH 6.0), but it is present in thiolsubtilisins and in the phenylboronic acid derivatives of the serine enzymes. The resonance was not visible in the mercuric or carboxamidomethyl derivatives of the thiol enzymes or in the phenylboronic acid--serine enzyme complex if the serine enzyme was first acylated with phenylmethanesulfonyl fluoride. The histidine at the catalytic site of thiolsubtilisin carries a positive charge between pH 5.6 and 8.4, in accord with previous data in favor of a mercaptide--imidazolium ion pair at the catalytic site. The charge distribution (- + -) at the active site of thiolsubtilisin and in the phenylboronic acid derivatives of the serine enzymes resembles that in the tetrahedral transition state formed between a serine enzyme and its substrate. Therefore, the stable hydrogen bond (found in the thiol enzyme and in the phenylboronic acid derivative of the serine enzyme) should be more important during catalysis than in the substrate-free enzyme.

On fait reagir le dicyclohexyl carbodiimide sur le O-phosphono-5 ribofuranosyle diphosphate pour obtenir le O-phosphono-5 ribofuranosyle phosphate-1,2. Une hydrolyse basique fournit les di-O-phosphono-2,5 ribofuranose et O-phosphono-5 ribofuranosyle phosphate