Nucleoside diphosphate kinase substrates

The subsequent cleavage of the thio-ester succinylCoA into succinate and coenzyme A by succinic acid-CoA ligase (succinyl CoA synthetase, succinic thiokinase) is strongly exergonic and is used to synthesize a phosphoric acid anhydride bond ( substrate level phosphorylation , see p. 124). However, it is not ATP that is produced here as is otherwise usually the case, but instead guanosine triphosphate (CTP). However, GTP can be converted into ATP by a nucleoside diphosphate kinase (not shown). 

ADP as a substrate in enzyme reactions, ADENYLATE KINASE (or MYOKINASE) ATP SYNTHASE CREATINE KINASE NUCLEOSIDE DIPHOSPHATE KINASE PHOSPHOGLYCERATE KINASE PYRUVATE KINASE RIBONUCLEOTIDE REDUCTASE SULFATE ADENYLYLTRANSFERASE (ADP) [ADP]/[ATP] ratio,... 

Jong, A.Y. Ma, J.J. Saccharomyces cerevisiae nucleoside-diphosphate kinase purification, characterization, and substrate specificity. Arch. Biochem. Biophys., 291, 241-246 (1991)... 

Williams, R.L. Oren, D.A. Munoz-Dorado, J. Inoue, S. Inoue, M. Arnold, E. Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution. J. Mol. Biol., 234, 1230-1247 (1993)... 

Schaertl, S. Konrad, M. Geeves, M.A. Substrate specificity of human nucleoside-diphosphate kinase revealed by transient kinetic analysis. J. Biol. Chem., 273, 5662-5669 (1998)... 

FIGURE 13-12 Ping-Pong mechanism of nucleoside diphosphate kinase. The enzyme binds its first substrate (ATP in our example), and a phosphoryl group is transferred to the side chain of a His residue. ADP departs, and another nucleoside (or deoxynucleoside) diphos-... 

The formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of a-ketoglutarate is a substrate-level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase (see Fig. 14-2). The GTP formed by succinyl-CoA synthetase can donate its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase (p. 505) ... 

Nucleoside diphosphates (NDP) are synthesized from the corresponding nucleoside monophosphates (NMP) by base-specific nucleoside monophosphate kinases (Figure 22.9). [Note These kinases do not discriminate between ribose or deoxyribose in the substrate.] ATP is generally the source of the transferred phosphate, because it is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in liver and muscle, where the turnover of energy from ATP is high. Its function is to maintain an equilibrium among AMP, ADP, and ATP. Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase—an enzyme that, unlike the monophosphate kinases, has broad specificity. 

Kandeel M, Kitade Y (2010) Substrate specificity and nucleotides binding properties of NM23H2/nucleoside diphosphate kinase homolog from Plasmodium falciparum. J Bioenerg Biomembr 42(5) 361-369... 

In some kinases, such as nucleoside diphosphate kinase, " an intermediate step is the phosphoryl transfer to a group belonging to the enzyme, as happens in ATPase and as was discussed in detail for alkaline phosphatase (Section V.B). In other kinases the phosphoryl transfer occurs directly from the donor to the acceptor in a ternary complex of the enzyme with the two substrates.Often metal ions like magnesium or manganese are needed. These ions interact with the terminal oxygen of the ATP molecule, thus facilitating the nucleophilic attack by the acceptor. The metal ion is often associated with the enzyme. For mechanistic schemes, see the proposed mechanism of action of alkaline phosphatase, especially when a phosphoryl enzyme intermediate is involved. 

McKenna reported the efiicient synthesis of the alpha, P-difluoromethylene deoxynucleoside 5 -triphosphate of adenosine and cytosine (89a,b), obtained enzymatically from catalytic ATP, phosphoenol pyruvate, nucleoside diphosphate kinase and pyruvate kinase using the nucleoside difluor-omethylene bisphosphonates as substrate for the kinase. The bispho-sphonate was prepared by nucleophilic displacement of the 5 -0-tosyl nucleoside precursor with tris(tetrabutylammonium) difluoromethylenebi-sphosphonate. Finally Mackman synthesised the diphosphate esters of two cyclopentyl based nucleoside phosphonates (90a,b). While (90b) was unstable following reduction of (90a) and therefore unsuitable for biological evaluation, (90a) exhibited potent inhibition against HIV reverse transcriptase. ... 

The nucleoside diphosphate kinase from human erythrocytes was of low specificity and would react with nucleoside triphosphates and nucleoside diphosphates which contained either ribose or deoxyribose and any of the natural purine or pyrimidine bases. The finding that alternative substrates, such as GTP and ATP, were competitive inhibitors, is consistent with the low substrate specificity... 

In summary, the nucleoside diphosphate kinase reaction is catalyzed by enzymes with broad substrate specificity many tissues show high activities of this enzyme, which is widely distributed in nature. 

Phosphorylation of the monophosphates is effected by a group of enzymes which are specific for the bases phosphorylation of the diphosphates is catalyzed by the ubiquitous nucleoside diphosphate kinase which has low specificity for both the base and pentose portions of its substrates. The latter enzyme occurs in animal cells in such high concentrations that the existence of the first step has been difficult to demonstrate in certain instances. 

The breakdown of succinyl coenzyme A may also be linked to the phosphorylation of GDP and IDP to yield GTP and ITP in the presence of inorganic phosphate. The enzyme catalyzing that reaction has been named the phosphorylating enzyme, and has been prepared in a crude form from heart muscle. This preparation also contains another enzyme, nucleoside diphosphate kinase, which catalyzes the transfer of phosphorus from GTP or ITP to ADP to yield ATP. The phosphorylation of ADP coupled to the oxidation of a-ketoglutarate is the only substrate level phosphorylation in the Krebs cycle, and, as can be expected, it is not inhibited by dinitrophenol. When O-labeled phosphate is used in the reaction, the label appears... 

However, nucleoside diphosphates (NDP) are still expensive substrates, which can be obtained from much more cheaper nucleoside monophosphates (NMP). In this respect we have combined the SuSy-catalyzed cleavage of sucrose with the enzymatic formation of NDPs from NMPs catalyzed by nucleoside monophosphate kinase (NMPK, EC 2.7.4.4) or myokinase (MK, EC 2.7.4.3), including in situ regeneration of ATP with pyruvate kinase (PK, EC 2.7.1.40) (Fig. 20) [272]. Testing the substrate spectrum of four different kinases disclosed that none of them accepted dTMP as substrate [272], However, dUMP was well accepted by NMPK and dUDP-activated glucose could also substitute dTDP-activated glucose as precursor for the synthesis of activated deoxysugars (see below). The excellent enzyme stabilities under synthesis... 

This enzyme activity is widely distributed in nature, and tissues generally show a higher concentration of this activity than of nucleoside monophosphate kinases. The enzyme has broad specificity purified preparations appear to have only the requirement that the substrates be a nucleoside triphosphate and a nucleoside diphosphate. 

The enzymatic machinery of the tricarboxylic acid cycle is located in a mitochondrial membrane system as mentioned above, the presence of nucleoside monophosphate and diphosphate kinases in this cell compartment enables the substrate-level phosphorylation associated with a-keto-glutarate oxidation to be coupled with the adenosine phosphates. 

The UDP-Gal epimerase system was also one of the early methods to employ pyruvate kinase (PK EC 2.7.1.40) for the conversion of UDP to UTP, with the simultaneous formation of pyruvate from phospho(enol)pyruvate (PEP). Previous kinase systems employed were either more expensive (nucleoside diphosphate kin-ase(EC 2.7.4.6)/ATP) or were thermodynamically less favorable (acetate kinase(EC 2.7.2.1)/acetyl phosphate). Another potentially usefid kinase system has recently been described. Noguchi and Shiba have utilized polyphosphate kinase (PPK) for the formation of UTP from UDP in a UDP-Gal recycling system [13]. Previously, PPK was shown to catalyze the reversible transfer of phosphate from ATP to ADP. Recently, it has been discovered that PPK will also accept other NDP and NTP substrates. For application to recycling systems, poly(phosphate) is more affordable than PEP as a phosphoryl donor. Synthetically, replacement of PK with PPK in the UDPGE-based recycling system efficiently provided LacNAc from GlcNAc on a multi-gram scale. 

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