scholarly journals The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5′[γ(S)-16O,17O,18O]triphosphate as substrate

1981 ◽  
Vol 199 (1) ◽  
pp. 227-233 ◽  
Author(s):  
G Lowe ◽  
B V L Potter
Keyword(s):  
Ternary Complex ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Enzyme Substrate ◽  
Gamma S ◽  
Yeast Hexokinase

Adenosine 5′[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an ‘in line’ mechanism.

scholarly journals The stereochemical course of phosphoryl transfer catalysed by glucokinase.

1982 ◽  
Vol 201 (2) ◽  
pp. 421-423 ◽  
Author(s):  
D Pollard-Knight ◽  
B V L Potter ◽  
P M Cullis ◽  
G Lowe ◽  
A Cornish-Bowden
Keyword(s):  
Rat Liver ◽  
Rate Constants ◽  
Ternary Complex ◽  
The Other ◽  
Chemical Mechanism ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Reaction Proceeds ◽  
Gamma S ◽  
Yeast Hexokinase

Adenosine 5'-[gamma(S)-16O,17O,18O]triphosphate has been used to determine the stereo-chemical course of phosphoryl transfer catalysed by rat liver glucokinase. The chirality of the product, D-glucose 6-[16O,17O,18O]phosphate was analysed by 31P n.m.r. spectroscopy. The reaction proceeds with inversion of configuration at phosphorus. The simplest interpretation of this result, which is the same as that observed with yeast hexokinase [Lowe & Potter (1981) Biochem. J. 199, 277-233], is that the phosphoryl group is transferred between MgATP2- and glucose in the ternary complex by an ‘in-line’ mechanism. It accords with the veiw that the kinetic differences between glucokinase and the other hexokinases arise from differences in rate constants and not from any fundamental differences in chemical mechanism.

Stereochemistry of phosphoryl transfer

1981 ◽  
Vol 293 (1063) ◽  
pp. 75-92 ◽  
Keyword(s):  
Absolute Configuration ◽  
Resonance Spectrum ◽  
Isotope Effects ◽  
Phosphate Esters ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Enzyme Substrate ◽  
Phosphate Monoesters ◽  
Line Transfer ◽  
General Method

A general method has been developed for the synthesis of chiral [ 16 O, 17 O, 18 O] phosphate monoesters of known absolute configuration. An analytic method for determining the absolute configuration of chiral phosphate esters has also been developed, which is based on the isotope effects of 17 O and 18 O at phosphorus in the 31 P nuclear magnetic resonance spectrum. These methods have shown that phosphoryl transfer catalysed by hexokinase, phosphofructokinase and pyruvate kinase occurs with inversion of configuration. This is most simply interpreted as an ‘in-line’ transfer of the phosphoryl group between substrates in the enzyme-substrate ternary complex.

scholarly journals The stereochemical course of phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal-muscle phosphofructokinase with a chiral [16O,17O,18O]phosphate ester

1981 ◽  
Vol 199 (2) ◽  
pp. 427-432 ◽  
Author(s):  
R L Jarvest ◽  
G Lowe ◽  
B V L Potter
Keyword(s):  
Skeletal Muscle ◽  
Phosphorus Atom ◽  
Bacillus Stearothermophilus ◽  
Ternary Complexes ◽  
Rabbit Skeletal Muscle ◽  
Phosphoryl Group ◽  
Phosphate Ester ◽  
Phosphoryl Transfer ◽  
Enzyme Substrate

Bacillus stearothermophilus and rabbit skeletal-muscle phosphofructokinases catalyse the transfer of the chiral [16O,17O,18O]phosphoryl group from D-fructose 1[(S)-16O,17O,18O],6-bisphosphate to ADP with inversion of configuration at the phosphorus atom. D-Fructose 1[(S)-16O,17O,18O],-bisphosphate was synthesized in situ from sn-glycerol 3[(S)-16O,17O,18O]phosphate. The simplest interpretation of these results is that the phosphoryl group is transferred between substrates in the enzyme substrate ternary complexes by an ‘in-line’ mechanism.

scholarly journals The stereochemical course of phosphoryl transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B)

1981 ◽  
Vol 199 (1) ◽  
pp. 273-276 ◽  
Author(s):  
R L Jarvest ◽  
G Lowe
Keyword(s):  
Escherichia Coli ◽  
Phosphorus Atom ◽  
Bacteriophage T4 ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Polynucleotide Kinase ◽  
Gamma S ◽  
Escherichia Coli B

Polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B) catalyses the transfer of the [gamma-16O,17O,18O]phosphoryl group from 5′[gamma(S)-16O,17O,18O]ATP to 3′-AMP with inversion of configuration at the phosphorus atom. The simplest interpretation of this observation is that the [gamma-16O,17O,18O]phosphoryl group is transferred directly from ATP to the co-substrate by an ‘in-line’ mechanism.

Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase fromSchizosaccharomyces pombe

1998 ◽  
Vol 76 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C Stan Tsai ◽  
Q Chen
Keyword(s):  
Pentose Phosphate Pathway ◽  
Ternary Complex ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Phosphoryl Group ◽  
Maximal Velocity ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Yeast Hexokinase

Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pombe have been purified 250-fold by an identical three-step. Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase. Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S. pombe (fission yeast). Hexokinase promotes Mg2+-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex. ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose. The Mg2+activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group. G6PDH catalyzes NADP+-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+as the leading reactant. High NADP+concentration inhibits the dehydrogenase by forming the dead-end ternary complex. In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP. Thus, the oxidative pentose phosphate pathway in S. pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP.Key words: yeast hexokinase, glucose-6-phosphate dehydrogenase.

scholarly journals Mechanistic insights into the phosphoryl transfer reaction in cyclin-dependent kinase 2: a QM/MM study

2019 ◽  
Author(s):  
Rodrigo Recabarren ◽  
Edison H. Osorio ◽  
Julio Caballero ◽  
Iñaki Tuñón ◽  
Jans Alzate-Morales
Keyword(s):  
Energy Barrier ◽  
Transfer Reaction ◽  
Theoretical Study ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Cyclin Dependent Kinase ◽  
Peptide Substrate ◽  
Bond Forming ◽  
Potential Energy Barrier ◽  
Charge Analysis

AbstractCyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results show that the base-assisted mechanism is preferred over the substrate-assisted pathway, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

scholarly journals Inhibition patterns of a model complex mimicking the reductive half-reaction of sulphite oxidase

1996 ◽  
Vol 319 (3) ◽  
pp. 953-959 ◽  
Author(s):  
Pradeep K CHAUDHURY ◽  
Samar K DAS ◽  
Sabyasachi SARKAR
Keyword(s):  
Steady State ◽  
Ternary Complex ◽  
Mixed Type ◽  
Catalytic Site ◽  
Binary Complex ◽  
Allosteric Site ◽  
Equilibrium Conditions ◽  
Enzyme Substrate ◽  
Sulphite Oxidase ◽  
Model Complex

Different inhibition types of the saturation kinetics involving a synthesized model complex, [Bu4N]2[MoVIO2(mnt)2] (E) (where mnt2- = 1,2-dicyanoethylenedithiolate), and HSO3- as the substrate (S) by structurally similar anions SO42-, H2PO4- and H2PO3- have been shown for the first time in relevance to the reductive half reaction of the native enzyme sulphite oxidase. SO42- acts as a competitive inhibitor. The mixed-type non-competitive inhibition by H2PO4- and the sigmoidal-type inhibition by H2 PO3- are explained by a diamond-configuration random-order model. This involves a random binding sequence of the substrate and the inhibitor, and forms, in addition to two binary complexes [enzyme-substrate (ES) and enzyme-inhibitor (EI)], one enzyme-substrate-inhibitor-type ternary complex (ESI) by participation of at least one more binding site in addition to the catalytic site. This is possible in the present case only by co-ordination enhancement of molybdenum in E. This co-ordination expansion is brought about by nucleophilic attack of the substrate or the inhibitor at the molybdenum, forming a hepta-coordinated binary complex with the generation of an oxoanionic functional site, called the allosteric site. Analysis of the experimental data suggests that the inhibition by H2PO4- is due to the mechanism following either equilibrium conditions or a combination of steady-state and equilibrium conditions. With H2PO3-, the inhibition is due to the mechanism following the steady-state conditions. It is also shown that the ternary complex involving the enzyme, substrate and H2PO4- or H2 PO3- is productive, but at a lower rate than that of the enzyme-substrate binary complex. Mixed-type inhibition with H2PO4- is actually of the type called ‘partially mixed competitive and non-competitive’ as the inhibitor binds both to the catalytic site and to the allosteric site. The sigmoidal-type inhibition by H2PO3- is similar to heterotropic allosteric effect of mixed V,K type with the distinction, however, that the significance of co-operativity in this case is of kinetic importance only. Received 3 January 1996/20 May 1996; accepted 25 June 1996

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