Environmental Effects on Phosphoryl Group Bonding Probed by Vibrational Spectroscopy:  Implications for Understanding Phosphoryl Transfer and Enzymatic Catalysis

2002 ◽  
Vol 124 (38) ◽  
pp. 11295-11306 ◽  
Author(s):  
Hu Cheng ◽  
Ivana Nikolic-Hughes ◽  
Jianghua H. Wang ◽  
Hua Deng ◽  
Patrick J. O'Brien ◽  
...  
Keyword(s):  
Vibrational Spectroscopy ◽  
Environmental Effects ◽  
Enzymatic Catalysis ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer

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 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 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 Structures of substrate- and nucleotide-bound propionate kinase fromSalmonella typhimurium: substrate specificity and phosphate-transfer mechanism

2015 ◽  
Vol 71 (8) ◽  
pp. 1640-1648 ◽  
Author(s):  
Ambika Mosale Venkatesh Murthy ◽  
Subashini Mathivanan ◽  
Sagar Chittori ◽  
Handanahal Subbarao Savithri ◽  
Mathur Ramabhadrashastry Narasimha Murthy
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Active Site ◽  
Structural Data ◽  
Transfer Mechanism ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Active Site Pocket ◽  
Bipyramidal Structure

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of L-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-boundSalmonella typhimuriumpropionate kinase are reported at 1.8–2.0 Å resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of ∼5 Å from the γ-phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occurviaan associative SN2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the β- and the γ-phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the γ-phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity.

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 Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

2012 ◽  
Vol 11 (10) ◽  
pp. 1249-1256 ◽  
Author(s):  
Matthew L. Fowler ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Entamoeba Histolytica ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Acetyl Phosphate ◽  
Phosphate Binding ◽  
Methanosarcina Thermophila ◽  
Inorganic Pyrophosphate ◽  
Content Type ◽  
Cell Extracts

ABSTRACTAcetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protistEntamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity inE. histolyticacell extracts in the direction of acetate/PPiformation but not in the direction of acetyl phosphate/Piformation suggests that the physiological direction of the reaction is toward acetate/PPiproduction. Kinetic parameters determined for each direction of the reaction are consistent with this observation. TheE. histolyticaPPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterizedMethanosarcina thermophilaATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for theM. thermophilaACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway inEntamoeba.

scholarly journals How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

2006 ◽  
Vol 70 (4) ◽  
pp. 939-1031 ◽  
Author(s):  
Josef Deutscher ◽  
Christof Francke ◽  
Pieter W. Postma
Keyword(s):  
Protein Phosphorylation ◽  
Sugar Transport ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Phosphotransferase System ◽  
Nitrogen And Phosphorus ◽  
Coupling Energy ◽  
Catalytic Function ◽  
Sugar Derivatives ◽  
Regulatory Functions

SUMMARY The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

scholarly journals Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase

2021 ◽  
Vol 77 (3) ◽  
pp. 357-368
Author(s):  
Ji Huang ◽  
Marie E. Fraser
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Citric Acid Cycle ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Specific Enzyme ◽  
Substrate Level ◽  
Mechanism Of Catalysis ◽  
Active Site Histidine ◽  
Succinyl Coa Synthetase

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

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