P.D. Boyer

Effect of temperature and antioxidants upon the lipoxidase-catalyzed oxidation of sodium linoleate.

Abstract The effects of temperature from −6 ° to 30 °C. and antioxidants upon the lipoxidase-catalyzed oxidation of aqueous sodium linoleate have been studied. An accurate analytical method for establishing the initial rates of the lipoxidase reaction has been described. The lipoxidase-catalyzed oxidation of linoleate was found to have a relatively low activation energy, 4300 cal./mole. Complete freezing of the lipoxidase system reduced the reaction rate to less than 1% of that found in the liquid system at the same temperature. All the phenolic antioxidants tested were found to inhibit the reaction. The greatest inhibitory effect was obtained with nordihydro-guaiaretic acid, followed closely by propyl gallate; the effective concentrations were sufficiently low so that these two antioxidants might prove useful in protecting foods from this type of oxidation. The biologically important antioxidant, α-tocopherol, was rapidly oxidized in the lipoxidase system.

Uses and limitations of measurements of rates of isotopic exchange and incorporation in catalyzed reactions.

Abstract Equations are given for the rate of enzymic reactions at equilibrium in terms of velocity constants and concentrations of enzyme and reactants. Relationships are developed for reactions of the type A ⇌ B and A + B ⇌ C + D. The general equation for the latter type may be used to derive relationships for simpler systems and for reactions involving compulsory order of combination of enzyme and substrates. The relationships show, among other things, that measurements of isotopic exchange rates at equilibrium can lead to determinations of dissociation constants for enzyme-substrate intermediates in some simple systems. The relationships may also be useful in detection of compulsory orders of combination of reactants with an enzyme. Equations for the relative rates of exchange of two reactants in an enzymic reaction define clearly conditions where these rates may be unequal and independent of steps in which covalent bonds are formed or broken. Limitations of exchange data to predict occurrence of reaction steps involving covalent bond formation are pointed out. In addition, the considerations impose severe and insufficiently recognized limitations on the measurements of the relative rates or amounts of isotope incorporation for prediction of the occurrence or importance of metabolic pathways.

Hypotheses for and some kinetic studies with glutamine synthetase and acetate thiokinase.

Abstract Kinetic studies with glutamine synthetase showed that the apparent Michaelis constant for glutamine was independent of the concentration of ammonia. Similarly, with other substrate combinations little or no interdependency of apparent Michaelis constants was found. Measurements of the rate of isotope exchange at equilibrium with glutamine synthetase showed that by variation of equilibrium conditions the rate of incorporation of glutamate into glutamine could be made considerably less than, equal, to or considerably greater than the rate of incorporation of inorganic phosphate into ATP. Likewise, with acetate thiokinase, variation of equilibrium conditions resulted in a rate of acetate incorporation into acetyl CoA which was approximately equal to or definitely greater than the rate of AMP incorporation into ATP. This demonstrates that with these enzymes, steps other than covalent bond forming and breaking steps can control equilibrium exchange rates. The results, however, limit mechanistic deductions from exchange data. Glutamine synthetase was found to require prior incubation with ATP for demonstration of maximum initial velocity. The significance of the findings are discussed in relation to alternative hypotheses for the glutamine synthetase and acetate thiokinase reactions, including a new hypothesis based on prior reaction of two substrates before reaction with ATP. Present information appears insufficient to allow a choice among various hypotheses.

The magnesium activation of pyrophosphatase.

Abstract The activity of rat brain pyrophosphatase at various concentrations of added Mg++ and inorganic pyrophosphate has been measured. In confirmation and extension of previous results the data show a pronounced inhibition of the enzyme action by pyrophosphate and a weaker inhibition by excess magnesium pyrophosphate. Concentrations of free and combined Mg++ and pyrophosphate in the assay conditions were calculated by use of an apparent dissociation constant for magnesium pyrophosphate as determined by spectrophotometric measurements of the competition of pyrophosphate and ethylenediamine tetraacetate for Mg++. Evidence is presented that the activation of rat brain pyrophosphatase by Mg++ can be ascribed to a requirement of the enzyme for magnesium pyrophosphate for a substrate. The requirement for a much higher Mg++ and pyrophosphate concentration for maximum activity under some conditions can logically result from the removal of the potent inhibition by free pyrophosphate and lack of inhibition by free Mg++.

On the enzymic synthesis of lactose-1-PO4.

Abstract Evidence is presented that enzyme fractions obtained from bovine mammary tissue will catalyze the synthesis of lactose 1-phosphate from UDPG and glucose 1-phosphate according to the reactions: UDPG UDPGal UDPGal + G-1-P 1-phosphate + UDP Using radioactive glucose, glucose 6-phosphate, and doubly labeled glucose 1-phosphate in the presence of UDPG and the proper fortifiers, it was shown that neither glucose nor glucose 6-phosphate would act as the galactosyl acceptor while glucose 1-phosphate would function as the acceptor. The phosphorylated sugar formed yielded equal amounts of lactose and inorganic phosphate upon mild acid hydrolysis and gave a negative reducing test in alkaline solution. It migrated more slowly than glucose 1-phosphate in paper electrophoresis and could be separated from glucose 1-phosphate by means of ion-exchange chromatography if the reaction medium was first treated with phosphoglucomutase to convert the glucose 1-phosphate to glucose 6-phosphate. When the lactose phosphate was enzymically synthesized from glucose 1-phosphate doubly labeled with C 14 and P 32 and UDPG, no radioactivity could be found in the galactose moiety. The glucose moiety of the lactose contained approximately the same specific activity as the substrate glucose 1-phosphate, and the ratio of the specific activities of C 14 to P 32 was the same in the glucose and phosphate of the lactose phosphate as in the starting glucose 1-phosphate. The primary nucleotide formed was identified as UDP by paper chromatography.

The biological inactivity of glucose 6-phosphite, inorganic phosphites and other phosphites☆☆☆

Abstract d -Glucose 6-phosphite has been synthesized by reaction of 1,2,3,4-tetra- O -acetyl-β- d -glucopyranose with PCl 3 followed by hydrolysis and deacetylation. d -Glucose 6-phosphite served neither as a substrate nor as an inhibitor of catalysis by hexokinase, glucose-6-phosphatase, glucose 6-phosphate dehydrogenase, or of fermentation by a yeast extract. Ethyl hydrogen phosphite, isopropyl hydrogen phosphite, and butyl hydrogen phosphite did not interact with intestinal phosphatase, and presumably cannot be hydrolyzed by this enzyme. Inorganic orthophosphite at 0.05 M inhibited intestinal phosphatase by 32%, but 0.01 M was without effect. Inorganic phosphite at 0.20 M inhibited 3-phosphoglyceraldehyde dehydrogenase slightly but nonspecifically; sodium sulfate at a comparable ionic strength produced a similar inhibition. At 0.067 M , phosphite had no influence on the capacity of washed yeast cells to ferment glucose, and at 0.02 M phosphites did not impair the respiration of an animal cell tissue culture. Phosphites showed a negligible or low order of toxicity for the rat. The inertness of other phosphites and other phosphite esters in biological reactions of phosphite or phosphate esters likely results from the differences in electrostatic charge distribution rather than of spatial arrangement of the phosphites and phosphates.

Orthophosphite as a buffer for biological studies

Abstract The second ionizable hydrogen of orthophosphorous acid was found to be 50% neutralized at pH 6.5 and an ionic strength of 0.08. Increase in temperature from 0 ° to 50 ° caused a pH increase of only 0.15 pH unit in 0.05 M phosphite buffer solution. Phosphite solutions were resistant to oxidation by molecular oxygen at temperatures up to 60 ° and over a pH range of 1.5–7.6, and were stable upon storage at room temperature. The salts CaHPO 3 and MgHPO 3 were considerably more soluble than the corresponding phosphate salts. The above properties of phosphite, together with their demonstrated inertness in a variety of biological reactions, invites consideration of phosphite as a buffer for biological studies in the pH range of 5.5–7.5.

The fate of oxygens of inorganic phosphate in photophosphorylation

Abstract In the formation of ATP from ADP and inorganic phosphate by photophosphorylation as catalyzed by chloroplasts, an oxygen is lost from the inorganic phosphate. Thus the photophosphorylation process involves either formation of a covalent phosphorylated intermediate, which seems most probable, or an activation such that an oxygen atom from inorganic phosphate is lost as the ATP is formed. The inorganic phosphate taken up appears almost exclusively in the terminal phosphoryl group of the ATP. The oxygen atoms of the inorganic phosphate, of the medium do not undergo any appreciable exchange with H 2 O oxygens when incubated with chloroplasts in the dark or in the light, and slight or no exchange occurs with the oxygens of inorganic phosphate incorporated into ATP.

Studies concerning the mechanism of action of acetoacetyl succinic thiophorase by use of O18.

Abstract In the reaction of succinate-O18 with acetoacetyl coenzyme A to form succinyl coenzyme A and acetoacetate, as catalyzed by acetoacetyl succinic thiophorase, O18 from the succinate appears in the carboxyl group of acetoacetate. An enzymic mechanism considered consistent with these findings involves formation of mixed anhydrides as intermediates.

The light-induced formation of rapidly phosphorylated compounds in chloroplasts

Abstract The demonstration of at least two rapidly formed phosphorylated compounds (presumably proteins) by phosphorylating spinach chloroplasts has been accomplished. Characterization of the more rapidly labeled compound shows it to be stable to alkaline dialysis and labile to acid dialysis. Migration of an alkaline digest of the phosphorylated chloroplast material during high-voltage electrophoresis suggests that it is neither protein-bound phosphohistidine nor protein-bound phosphoserine. Ammonium chloride (4 × 10 −3 M ) inhibits both the formation of the phosphorylated compound and the total formation of ATP. Trace amounts of ADP (4.5 μ M ) are needed for maximal formation of the protein-bound phosphate. Either phenazine methosulfate or flavin mononucleotide enhances incorporation of P i 32 into the protein-bound phosphate fraction. Addition of either excess ADP or excess ATP to the reaction mixture reduces the incorporation of P i 32 into the protein-bound fraction. The possibility that this compound represents a phosphorylated intermediate in photosynthetic phosphorylation is discussed.