Phosphoryl transfer mechanisms

ATP + (d)CMP = ADP + (d)CDP (<4> formation of a ternary complex, addition of substrates is random [5] <1> reaction proceeds by a sequential mechanism, a ternary complex of the enzyme with both substrates is formed as the central intermediate in the reaction [12] <3> reaction mechanism is sequential and nonequilibrium in nature, substrates bind to the enzyme in a random order, substrate binding is cooperative [14] <7> the mechanism is analogous to the phosphoryl transfer mechanism in cAMP-dependent protein kinase that phosphorylates the hydroxyl groups of serine residues [16] <8> random bi-bi mechanism [17])... 

In order to solve this mechanistic problem, a method for analysing the stereochemical course of phosphokinases has been developed using chiralf160,170,180]phosphate esters. Stereochemical analysis should allow a clear mechanistic distinction to be made, since inversion of configuration at phosphorus implies a direct in-line phosphoryl transfer mechanism whereas retention of configuration suggests a doubledisplacement mechanism with a phosphoryl-enzyme intermediate on the reaction pathway. 

Other phosphoryl transfer mechanisms are an associative, two-step mechanism (An + Dn) and a concerted mechanism (ANDN) with no intermediate. The AN+DN mechanism is an addition-elimination pathway in which a stable pentacoordinate intermediate, called a phosphorane, is formed. This mechanism occurs in some reactions of phosphate triesters and diesters, and has been speculated to occur in enzymatic reactions of monoesters. In the concerted ANDN mechanism, bond formation to the nucleophile and bond fission to the leaving group both occur in the transition state. This transition state could be loose or tight, depending upon the synchronicity between nucleophilic attack and leaving group departure. The concerted mechanism of Fig. 2 is drawn to indicate a loose transition state, typical of phosphate monoester reactions. 

An example of a random, single-displacement mechanism is seen in the enzyme creatine kinase, a phosphoryl-transfer enzyme that uses ATP as a phosphoryl... 

FIGURE 19.29 A mechanism for the pyruvate kinase reaction, based on NMR and EPR studies by Albert Mildvan and colleagues. Phosphoryl transfer from phosphoenolpyrnvate (PEP) to ADP occurs in four steps (a) a water on the Mg ion coordinated to ADP is replaced by the phosphoryl group of PEP (b) Mg dissociates from the -P of ADP (c) the phosphoryl group is transferred and (d) the enolate of pyruvate is protonated. (Adapted from Mildvan, A., 1979. Advances in Eiizymology 49 103-126.)... 

Two phosphoribosyl transferases then convert adenine to AMP and hypoxanthine and guanine to IMP or GMP (Figure 34-4). A second salvage mechanism involves phosphoryl transfer from ATP to a purine ri-bonucleoside (PuR) ... 

For acyl transfer, phosphoryl transfer, and sulfonyl transfer, the primary kind of evidence in favor of concerted mechanisms for some reactions is a linear Bronsted plot of log k versus for a range of nucleophiles, spanning p/ir/ = - p r s = 0, coupled... 

Compound (32) phosphorylates hindered alcohols in the presence of EtOH consistent with a dissociative mechanism involving a metaphosphate-like intermediate,since it proceeds with considerable racemization at phosphorus(Scheme 6). The extent of phosphoryl transfer which proceeds with retention of configuration is ca. 357, (i e. ca. 707. racemization) the excess of (S)p configuration would arise from transfer with configurational inversion, and might indicate a relatively free ... 

In the case of La3 + - and Zn2+-catalyzed methanolysis of the phosphorothioate esters the observed / ig values of —0.87 and —0.74 also signify an associative mechanism with some departure of the leaving group, but it is difficult to assign the extent of the bond cleavage since the /ieq value is not known for the phosphoryl transfer between thiol and oxygen nucleophiles. 

Chen, G., Porter, M.D., Bristol, J.R., Fitzgibbon, M.J., and Pazhanisamy, S., Kinetic mechanism of the p38-alpha MAP kinase phosphoryl transfer to synthetic peptides, Biochemistry, 39, 2079, 2000. 

This phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form ATP Aeq = [ATP][acetate]/ [acetyl phosphate] [ADP] = 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzyme-bound acyl-phosphate intermediate, but later kinetic and stereochemicaT data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzyme-bound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively. 

However, for E-P formation from E-Nas with 1.0 mM ATP and 2.0 mM ADP, observed = 4.2 X 10 s indicating that phosphoryl transfer from bound ATP to the enzyme is not rate-limiting for E-P formation from E-Nas. Their results suggest that there is likely to be a rate-limiting conformational change of the E-Nas-ATP intermediate, followed by rapid phosphoryl transfer, with kcat = 3000 s See Membrane Transport Binding Change Mechanism... 

FIGURE 19. The two- and three-metal-ion mechanisms for phosphoryl transfer in the group I introns. Oxygens in bold have been identified as metal ligands by ion rescue experiments. From Reference 177. Reprinted with permission from AAAS... 

Schneider, B. Babolat, M. Xu, Y.W. Janin, J. Veron, M. Deville-Bonne, D. Mechanism of phosphoryl transfer by nucleoside diphosphate kinase pH dependence and role of the active site Lysl6 and Tyr56 residues. Eur. J. Biochem., 268, 1964-1971 (2001)... 

Although this reaction is fully reversible, the relatively high [ATP]/[ADP] ratio in cells normally drives the reaction to the right, with the net formation of NTPs and dNTPs. The enzyme actually catalyzes a two-step phosphoryl transfer, which is a classic case of a double-displacement (Ping-Pong) mechanism (Fig. 13-12 see also Fig. 6-13b). First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme... 

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