Acetate kinase

Acetate kinase is phosphorylated by acetyl phosphate and it has been shown that the phosphoenzyme can synthesise ATP from ADP, and acetyl phosphate from acetate. The mode of decomposition of carbamyl phosphate in aqueous solution is pH dependent and can proceed with either the production of ammonia and carbon dioxide (equation 1), or cyanate (equation 2). No cyanate could be detected during the hydrolysis... 

A tri-enzymatic sensing layer based on kinase-oxidase activities for the detection of acetate was also described. A reaction sequence using acetate kinase, pyruvate kinase and pyruvate oxidase enabled the production of H2O2 in response to acetate injection in the range 10 pM - 100 mM59. 

Adenosine triphosphate (ATP) is one of the most important cofactors involved in many of the synthetic reactions going on within the cell. Its recent large scale in vitro enzymatic synthesis from adenosine and acetylphosphate is of particular interest. Three enzymes immobilized in polyacrylamide gel were used adenosine kinase, adenylate kinase and acetate kinase (lip. ... 

Figure 11.4. The active site of acetate kinase. Reprinted with permission from Buss et al. (2001), copyright 2001 American Society for Microbiology.

Buss KA, Cooper DR, Ingram-Smith C, et al. 2001. Urkinase structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases. J Bacte-riol 183 680-6. 

Ingram-Smith C, Barber RD, Ferry JG. 2000. The role of histidines in the acetate kinase from Methanosarcina thermophila. J Biol Chem 275 33765-70. 

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. 

An enzymatic reaction intermediate formed by phospho-ryl transfer to a carboxyl group on an enzyme. Acyl-phosphates are structurally analogous to acid anhydrides (R—CO —O —CO—R ), and they are thermodynamically less stable than either of the two phosphoanhydride bonds in ATP. This is evident by the fact that the acetate kinase reaction (ADP + acetyl-phosphate = ATP + acetate) favors ATP formation with an equilibrium constant of about 3,000. Acetyl-phosphate can be chemically synthesized by reacting orthophosphate with acetic anhydride. 

This procedure involves the enzymatic conversion of the chiral acetate (C(HDT)COO ), obtained from experiments involving chiral methyl groups, to labeled malate. The enzymes used in this procedure include acetate kinase, phosphotransacetylase, malate synthase, and fumarase. 

Occasionally, one may also wish to use an auxiliary enzyme not as an assay system but strictly as a means for maintaining the steady-state concentration of a primary reactant in a multisubstrate reaction system. For instance, acetate kinase (and its substrate acetyl phosphate) or creatine kinase (and its substrate creatine phos-... 

Occasionally, one can maintain initial rate conditions by using a coupled reaction system to regenerate one of the limiting substrates. For example, to regenerate ATP in a phosphotransferase reaction, one can use creatine phosphate and creatine kinase acetylphosphate and acetate kinase or phosphoenolpyruvate and pyruvate kinase. 

ACETATE KINASE. MacNeal et first proposed the use of Escherichia coli acetate kinase as a nearly ideal... 

For most reactions, one can use 1-2 mM acetyl phosphate and 1-2 international units of acetate kinase. The enzyme is usually supplied as a crystalhne suspension in 3 M ammonium sulfate, and 10 mM ammonium ion is inhibitory. Therefore, a useful practice is to snip off 0.5 cm from a disposable Eppendorf micropipette tip to facih-tate removal of 10-20 microliters of the crystalline suspension then spin down the enzyme in a 1.5 ml disposable conical plastic centrifuge tube, and remove the ammonium sulfate solution with a wick of twisted Kim-wipe. The enzyme precipitate can now be taken up directly into your working buffer. Note Acetate kinase is inactivated by cold exposure, but incubation with 10 M ATP or GTP reactivates the enzyme if warmed to room temperature for 5-10 min. 

LACTATE MONOOXYGENASE LEUCINE KINETICS ACETATE KINASE... 

ACETATE KINASE (PYROPHOSPHATE) Acetate kinase reduction,... 

ACETATE KINASE ACETYL-CoA CARBOXYLASE ACETYL-CoA SYNTHETASE N-ACETYLCLUCOSAMINE KINASE ACTIN ATPase ACTOMYOSIN ATPase N-ACYLMANNOSAMINE KINASE ADENINE NUCLEOTIDE TRANSLOCASE ADENOSINE KINASE ADENYLATE KINASE (MYOKINASE) ADENYLYLSULFATE KINASE d-ALANINE-d-ALANINE LIGASE... 

Orthophosphate as substrate or product, ACETATE KINASE (PYROPHOSPHATE) ACETYL-CoA CARBOXYLASE ACID PHOSPHATASE ACTOMYOSIN ATPase ACYL PHOSPHATASE ASPARTATE-SEMIALDEHYDE DEHYDROGENASE ATPases... 

The multi-enzyme system was recently utilized for the synthesis of dTDP-4-keto-6-deoxy-a-D-glucose 3 and dTDP-P-L-rhamnose 5 generating dlTP from dTMP by dTMP kinase and acetate kinase [66, 71]. Further combination with GerB, a dTDP-4-keto-6-deoxy-D-glucose aminotransferase, gave dTDP-4-amino-4,6-dideoxy-D-glucose [72]. 

ATP Triphosphate Chain Conformation. Much of the work in the area of ATP triphosphate chain conformation has been performed by Cleland and co-workers (14--16). Their studies on metal(III)ATP interactions with kinases have led to the classification of kinases according to the stereochemistry of the polyphosphate chain as it binds to the active site. For the kinases they studied (hexokinase, glycerokinase, creatine kinase, phosphofructokinase, 3-phosphoglycerate kinase, acetate kinase, arginine kinase, adenylate kinase and pyruvate kinase) it was found that B, y-bidentate chromi M(III)-ATP (CrATP) and not a,6,y-tridentate CrATP is a... 

For ATP regeneration, a similar concept has been used (Berke et al, 1988). ATP can be regenerated from ADP using acetyl phosphate and the enzyme acetate kinase, upon release of acetate. The reaction is irreversible and acetyl phosphate is a relatively cheap phosphate donor. Thus, in an enzyme membrane reactor, PEGderivatized ATP was consumed by a phosphorylase or a synthetase in a reaction leading to a product of interest, and the ATP was regenerated by the acetate kinase (Figure 10.9). 

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