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H Hoving

Publications and source records attributed to H Hoving.

8 recordsLinked to original sources
PubMed1985

Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I.

The stereochemistry of the transcarboxylase-catalyzed carboxylation of 3-fluoropyruvate has been studied by using fluorine NMR of unpurified reaction mixtures. When the product 3-fluorooxaloacetate was trapped by using malate dehydrogenase, only the 2R,3R diastereomer of 3-fluoromalate was formed. The fluoromethyl group of fluoropyruvate does not take up deuterium label from the solvent during the reaction. These results confirm and extend those obtained previously by Walsh and co-workers [Goldstein, J. A., Cheung, Y. F., Marletta, M. A., & Walsh, C. (1978) Biochemistry 17, 5567-5575] showing that transcarboxylase is specific for one of the two prochiral hydrogens in fluoropyruvate. Transcarboxylase, coupled to malate dehydrogenase, has been used to analyze samples of chiral fluoropyruvate obtained by dephosphorylation of (Z)-fluorophosphoenolpyruvate in D2O in the presence of either pyruvate kinase or enzyme I from the Escherichia coli sugar transport systems. Analysis of the fluoromalate produced showed that fluoroenolpyruvate is deuterated from opposite faces by these two enzymes: enzyme I protonates (deuterates) fluoroenolpyruvate exclusively from the 2-re face and pyruvate kinase does so mainly from the 2-si face. Fluoropyruvate is carboxylated by transcarboxylase with absolute retention of configuration.

Animals↗
PubMed1983

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: stereospecificity of proton transfer in the phosphorylation of enzyme I from (Z)-phosphoenolbutyrate.

The stereochemistry of the proton transfer in the reaction of phosphoenolbutyrate with enzyme I has been established. During the reaction of the pure Z isomer of this analogue of phosphoenolpyruvate with enzyme I, to yield phosphoenzyme I and 2-oxobutyrate, the substrate is protonated at C-3 from the 2re,3si face. This stereospecificity was established for the transfer of a proton to (Z)-phospho[3-D]enolbutyrate and for the transfer of a deuteron to (Z)-phospho[3-H]enolbutyrate. The E isomer of phosphoenolbutyrate is not a substrate for enzyme I. Accordingly, the reaction of phosphoenzyme I with 2-oxobutyrate yields exclusively the Z isomer of phosphoenolbutyrate, and only the pro-S proton at C-3 of 2-oxobutyrate is abstracted. A kinetic H/D isotope effect of 6.8 in this reaction demonstrates the rate-limiting nature of the proton-transfer step. The stereochemical analysis of 2-oxo[3(R)-H,D]butyrate and of 2-oxo-[3(S)-H,D]butyrate was carried out by using the pyruvate kinase catalyzed enolization of this compound. This enzymatic enolization, with phosphate as a cofactor, is rapid at neutral pH and is a highly stereospecific reaction: only the pro-R proton at C-3 of 2-oxobutyrate is exchanged with solvent. This reaction was also used to generate the pure 3R and 3S enantiomers of 2-oxo[3-H,D]butyrate. The degree of protonation/deuteration at C-3 of 2-oxobutyrate was detected from the fine structure of the methyl proton nuclear magnetic resonance signal.

Escherichia coli↗
PubMed1982

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: role of divalent metals in the dimerization and phosphorylation of enzyme I.

The function of divalent metal ions (Mg2+ and Mn2+) in the dimerization and phosphorylation of enzyme I has been studied. Only a dimeric form of the enzyme can be phosphorylated [Misset, O., Brouwer, M., & Robillard, G. T. (1980) Biochemistry 19, 883--890; Hoving, H., Lolkema, J. S., & Robillard, G. T. (1981) Biochemistry 20, 87--93]. Kinetic studies of phosphoryl-group exchange between phosphoenolpyruvate and pyruvate and measurements of initial enzyme I phosphorylation rates revealed that a divalent metal ion must be bound to the enzyme to render the dimer active. Mn2+ binding experiments by means of electron paramagnetic resonance showed binding of at least one Mn2+ per unphosphorylated dimer with a binding constant comparable to the activation constant found in the kinetic studies and a 10-fold tighter binding of only one Mn2+ per phosphorylated dimer. Gel filtration experiments provided evidence that divalent metals produce about a 10-fold stabilization of the dimers, in addition to their effect on the specific dimer activity. The stability of the dimer was also strongly dependent on salts such as LiCl, NaCl, KCl, and a series of tetraalkylammonium chlorides. The relative effects of these salts suggest that hydrophobic interactions possibly play a significant role in enzyme I dimerization.

Escherichia coli↗
PubMed1981

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: equilibrium kinetics and mechanism of enzyme i phosphorylation.

The phosphorylation of enzyme I from the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system was studied by means of isotope exchange between phosphoenolpyruvate and pyruvate. Experiments monitoring 1H--2H exchange showed that enzyme I phosphorylation is accompanied by the transfer of a proton from the enzyme to the C-3 atom of the substrate. 14C--12C-exchange experiments with both deuterated and protonated pyruvate exhibited a kinetic isotope effect (nu V/nu D = 1.9), showing that the proton transfer is (partly) rate determining and is an essential step in the mechanism of phosphoryl group transfer. Under certain reaction conditions, a more than proportional increase of the 14C exchange rate with increasing total enzyme concentration was observed, indicating that only the dimeric form of enzyme I is phosphorylated. From the dependence of the 14C exchange rate on the phosphoenolpyruvate and pyruvate concentrations, the forward and reverse second-order rate constants of the reaction were determined to be 3 X 10(7) and 8 X 10(5) M-1 min-1, respectively, yielding an equilibrium constant of approximately 40 and a delta G degree for enzyme I phosphorylation of --2.3 kcal/mol. The significance of the values of these rate constants for the thermodynamics of the phosphotransferase system is discussed.

Binding Sites↗
PubMed1977

Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site.

The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.

Animals↗
PubMed1977

Transcriptional units for ribosomal proteins in yeast.

The effects of ultraviolet irradiation on the rates of synthesis of individual ribosomal proteins in yeast were examined and compared with the ultraviolet sensitivities of the synthesis of other yeast proteins. It was found that the synthesis of yeast ribosomal proteins is much more sensitive to ultraviolet irradiation than that of other yeast cellular proteins. Taking into account the half-life of yeast mRNA, the results obtained indicate that the genes coding for ribosomal proteins form part of long transcriptional units, which are much longer than the DNA stretch needed to code for a ribosomal protein of average molecular weight. Saturation hybridization of total poly(A)-containing mRNA with yeast nuclear DNA revealed that as much as 30% of DNA is complementary to yeast mRNA. Thus, the primary transcript of a protein gene on the average is about 1.7 times the length of the actual messenger. On the basis of the various experimental data we suggest a clustering of the yeast ribosomal protein genes in a number of common transcriptional units.

DNA↗