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Biomedical subjects

J R Knowles

Publications and source records attributed to J R Knowles.

At least 145 records · Page 8Linked to original sources
PubMed1977

Phosphoglycerate mutase from wheat germ: studies with 18O-labeled substrate, investigations of the phosphatase and phosphoryl transfer activities, and evidence for a phosphoryl-enzyme intermediate.

From studies using unlabeled phospho-D-glycerate in solutions enriched in H2(18)O, and from experiments involving [18O]phospho-D-glycerate, it is shown that the intramolecular isomerization of 2- and 3-phospho-D-glycerate that is catalyzed by the phosphoglycerate mutase from wheat germ does not involve an intermediate 2,3-cyclic phosphate. It is also shown that phosphoglycerate mutase catalyzes the hydrolysis of the substrate analogues 2-phosphoglycolate, 2-phospho-D-lactate, 3-phosphohydroxypropionate, phosphoenolpyruvate, and phosphohydroxypyruvate. The substrates 3- and 2-phospho-D-glycerate are not hydrolyzed, nor are 2,3-bisphospho-D-glycerate, 2-phospho-L-lactate, 3-phospho-L-glycerate, or sn-glycerol 3-phosphate. Although no exchange of D-[14C]glycerate into phospho-D-glycerate can be detected, the enzyme catalyzes the transfer of the phosphoryl group from "unnatural" donors such as 2-phosphoglycolate, to the "natural" acceptor, D-glycerate. It is concluded that the intramolecular phosphoryl transfer catalyzed by the wheat germ phosphoglycerate mutase follows a pathway involving a phosphoryl-enzyme intermediate.

Glycerophosphates↗
PubMed1977

To stabilize a transition state.

Inspection of the active sites of the many enzymes whose structures are known at high resolution leads to the unsurprising conclusion that an enzyme may provide an environment that exquisitely stabilizes the transition state for an elementary catalytic step that is expected to be difficult. In an effort both to mimic such an environment and to have the opportunity of investigating the thermodynamic and kinetic consequences of juxtaposing polar and non-polar loci in the same molecule, we have synthesized several specifically functionalized alpha-cyclodextrins. One of these is designed to stabilize the trigonal bipyramidal transition state for an in-line displacement at the phosphorus of a phosphate monoester. This cyclodextrin contains three symmetrically disposed ammonium groups on the 'top' (at C-6) of the hydrophobic cavity formed by the hexa-glucose torus and the remaining 15 hydroxy groups are methylated. The thermodynamic consequences of adjacent hydrophobic and hydrogen-bonding and/or electrostatic binding sites are investigated using several charged and uncharged ligands. The feasibility of building host species explicitly to stabilize reaction transition states is discussed.

Dextrins↗
PubMed1976

Deuterium and tritium exchange in enzyme kinetics.

The theory of the isotopic exchange of deuterium and tritium between an enzyme-substrate complex and the solvent is derived for 16 different types of experiment involving measurements of initial velocities and of the isotopic content of the reactants and products as a function of the extent of reaction. It is shown how the data from these experiments can be analyzed to obtain the rate constants for the individual steps and thereby the Gibbs free energies of the intermediates and transition states in the reaction. The effects of isotopic substitution on each intermidiate and transition state are also found and this allows conclusions to be drawn as to the extent to which a proton is in flight in a particular transition state. Neither substrate handling (that is, on-off steps), nor the isotopic exchange with the solvent, is assumed to be rapid.

Deuterium↗
PubMed1976

Energetics of triosephosphate isomerase: the appearance of solvent tritium in substrate dihydroxyacetone phosphate and in product.

When the isomerization of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is catalyzed by triosephosphate isomerase in tritiated water, both the substrate and product become labeled. The specific radioactivity of the product is about 80% that of the solvent, which shows that the protonation of the enediol intermediate at C-2 (to form the enzyme-bound product D-glceraldehyde 3-phosphate) is followed by a slower step not involving proton transfer. The specific radioactivity of the remaining substrate after partial reaction rises as the reaction proceeds and shows that the reaction intermediate that exchanges protons with the medium returns to dihydroxyacetone phosphate (picking up tritium) about one-third as often as it is converted to D-glceraldehyde 3-phosphate. These results allow a qualitative description of the relative heights of the energy barriers in the catalyzed reaction and contribute to the quantitative analysis of the energetics of the process.

Carbohydrate Epimerases↗
PubMed1976

Energetics of triosephosphate isomerase: the appearance of solvent tritium in substrate glyceraldehyde 3-phosphate and in product.

When the isomerization of D-glyceraldehyde 3-phosphate to dihydroxyacetone phosphate is catalyzed by triosephosphate isomerase in tritiated water, both the substrate and the product become labeled. The specific radioactivity of the product is only about 13% that of the solvent, which shows that the protonation of the enediol intermediate at C-1 (to form the enzyme-bound product dihydroxyacetone phosphate) is a kinetically significant step, and that the rate of loss of dihydroxyacetone phosphate from the enzyme is relatively fast. The specific radioactivity of the remaining substrate after partial reaction rises as the reaction proceeds and shows that the reaction intermediate that exchanges protons with the medium returns to D-glyceraldehyde 3-phosphate about one-third as often as it is converted to dihydroxyacetone phosphate. These results confirm the qualitative description of the relative heights of the energy barriers in this reaction and further contribute to the quantitative analysis of the free-energy profile.

Carbohydrate Epimerases↗
PubMed1976

Energetics of triosephosphate isomerase: deuterium isotope effects in the enzyme-catalyzed reaction.

The effect of isotopic substitution of the specifically labilized hydrogen in the substrates of triosephosphate isomerase on the steady-state rates of the enzyme-catalyzed reaction has been examined. The k cat value for the enzyme-catalyzed transformation of [1(R)-2H] dihydroxyacetone phosphate is 2.9 times smaller than that for the 1(R)-1H compound. Because of the rapid loss of 2H to solvent from the enzyme-enediol complex, this factor represents the full kinetic isotope effect of the proton abstraction step. The values of k cat and of Km for D-[2-2H]glyceraldehyde 3-phosphate are indistinguishable from those of the 2-1H material. This arises from the rapid loss of 2H from the enzyme-enediol intermediate, which results in 1H rather than 2H transfer in the rate-limiting step. The steady-state kinetic results reported in this paper qualitatively confirm and quantitatively extend the results from the previous papers on the variation of the free energy along the reaction path.

Carbohydrate Epimerases↗
PubMed1976

Energetics of triosephosphate isomerase: the nature of the proton transfer between the catalytic base and solvent water.

The isomerization of specifically deuterium-labeled [1(R)-2H5dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, catalyzed by the enzyme triosephosphate isomerase, has been studied. It is shown that the extent of transfer of the 2H label from the substrate to the product D-glyceraldehyde 3-phosphate is (after complete reaction) the same as that of the corresponding transfer of 3H. The absence of an isotope effect shows that the exchange process of the tstopically labeled enzyme carboxyl group, -COOL H2O leads to -COOH + LOH, does not tnvolve a rate-limiting transition state in which L is the flight. Possible modes for the nature of the ionization of -COOL in 1H2O are discussed.

Carbohydrate Epimerases↗
PubMed1976

Free-energy profile of the reaction catalyzed by triosephosphate isomerase.

The experimental results on the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate catalyzed by triosephosphate isomerase that are presented in the previous five papers are here collected and analyzed according to the theory presented in the first paper (Albery, W.J., Knowles, J.R. (1976), Biochemistry 15, the first of eight papers in a series in this issue). The rate constants and fractionation factors so derived allow the construction of theGibbs free-energy profile for this enzyme-catalyzed reaction.

Carbohydrate Epimerases↗
PubMed1976

The "phosphoryl-enzyme" from phosphoglycerate kinase.

The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably just a tight complex between 1,3-bisphosphoglycerate and the enzyme. The two partial exchange reactions (between adenosine 5'-diphosphate, and adenosine 5'-triphosphate, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are very much slower than the rate of overall catalysis. No substrate analogue was found that accelerated the partial exchange reactions. Catalysis of each of the two exchange reactions and of the kinase reaction coincides after isoelectric focusing of purified enzyme, but the amount of cosubstrate necessary to cause the observed partial exchange rates is so small that these reactions may well be artifactual. The balance of evidence does not support a ping-pong pathway via phosphoryl-enzyme, and the reaction may be a sequential one in which the phosphoryl group is transferred between substrates in a ternary complex. The results point to the dangers in the interpretation of experiments where very small amounts of contaminating cosubstrate can lead to large kinetic effects, and to the possibility of mistaken deductions about the identity of reaction intermediates.

Adenosine Diphosphate↗
PubMed1976

Crystalline 3-phospho-d-glycerate kinase from horse muscle.

Phosphoglycerate kinase has been isolated in crystalline form from horse muscle. A convenient isolation procedure is described that yields homogeneous enzyme of specific activity 700 units/mg (30 degrees C). The enzyme is monomeric, and has a molecular weight 47 000. Of the eight cysteine residues in the protein, two react rapidly with Nbs21 with the concomitant loss of the catalytic activity. Since the isolation of phosphoglycerate kinase from yeast (Bücher, 1955) there have been several reports of purification methods yielding enzyme approaching molecular homogeneity, from rabbit muscle (Beisenherz et al., 1953; Czok and Bücher, 1960; Rao and Oesper, 1961; Avramov and Repin, 1965; and Scopes, 1969) and from chicken muscle (Gosselin-Rey, 1965). Crystalline material has been isolated from human erythrocytes (Hashimoto and Yoshikawa, 1962), and from yeast and rabbit muscle (Krietsch and Bücher, 1970). Cystallographic work on phosphoglycerate kinase from horse muscle by Blake et al. (1972) and Blake and Evans (1974) has prompted mechanistic interest in the enzyme, and we report here a simplified isolation procedure and some properties of the crystalline material from this source.

Amino Acids↗
PubMed1976

The intrinsic pKa-values of functional groups in enzymes: improper deductions from the pH-dependence of steady-state parameters.

The assumptions implicit in the deductions made from the pH-dependence of rate measurements of enzyme-catalyzed reactions are summarized, and the limitations of such determinations are discussed nonalgebraically. The following types of pH-profile are considered (in order of increasing utility): pH-"activity" curves at fixed [S] o;pH-dependences of kcat, Km, kcat/Km, and Ki; pH-dependences of kmodification (by specific reagents) and of competitive labeling (by nonspecific reagents); pH-dependences of elementary steps; and the direct observation of a titrating group.

Enzymes↗
PubMed1975

The orientation and accessibility of substrates on the active site of triosephosphate isomerase.

Tritiated sodium borohydride was used to reduce the substrates of triosephosphate isomerase in the presence of the enzyme, and the mixture of the four possible products (D-[1(R)-3H]; D-[1(S)-3H]-; D-[2-3H]-, and L-[2-3H]glycerol 3-phosphate) was analyzed. While enzyme-bound dihydroxyacetone phosphate is reduced completely stereoselectively and at a rate eight imes faster than in free solution, D-glyceraldehyde 3-phosphate is inaccessible to reduction by borohydride when bound to the active site of the enzyme.

Animals↗