Mg-ATP complex

Suzuki et al. examined the effect of various divalent cations on purified recombinant human GCH expressed in Escherichia coli to clarify the molecular mechanism of action of divalent cations on the GCH enzymatic activity [150]. They demonstrated that GCH utilizes metal-free GTP as the substrate for the enzyme reaction. Inhibition of the GCH activity by divalent cations such as Mg(II) and Zn(II) was due to a reduction in the concentration of metal-free GTP substrate by complex formation. Many nucleotidehydrolyzing enzymes such as G proteins and kinases recognize Mg-GTP or Mg-ATP complex as their substrate. In contrast with these enzymes, Suzuki et al. demonstrated that GCH activity is dependent on the concentration of Mg-free GTP [150]. 

A central question in phosphotransferases and nucleotidyltransferases is the structure of the metal-nucleotide complex which is the true substrate for the enzyme. It is unlikely that all of the possible Mg-ATP complexes could serve as substrates for a given enzyme, but until recently there has been no way to determine which isomer is active. The difficulty is the coordination exchange equilibrium, which is rapidly set up and dynamically maintained in solutions of Mg-ATP. To avoid this problem, metal-nucleotide complexes have been synthesized using coordination exchange-inert metals such as Cr(III) and Co(IIl) in place of Mg(II) [7,60], The resulting complexes are structurally stable and can be separated by chromatographic methods into their coordination isomers and stereoisomers. The isomers can then be investigated as substrates or inhibitors of specific enzymes. 

Magnesium is distinguished by the fact that it is required by most ATP-using enzymes. Here, Mg occurs as a complex with ATP, as shown in Figure 10,49. In other words, the true substrate for most ATF-requiring enzymes is not ATP, but the Mg-ATP complex. A deficiency in magnesium is uncommon. When it does occur, the physiological funchon that is most sensitive is neuromuscular activity. In molecular terms, the enzymes involved in neuromuscular activity that appear to be sensitive to Mg deficiency are those involved in the transport of sodium, potassium, and calcium, these enzymes are Na.K-ATPase and the calcium pump (Ca-ATPase). 

Most or all ATP-requiring enzymes use ATP in the form of the Mg-ATP complex. ATP chelates the magnesium ion, which is a divalent metal ion. The glycolytic pathway features a number of Mg-requiring enzymes. One of these enzymes is phosphofructokinase. The Mg requirement for this enzyme is illustrated by the data in Figure 10-50- Ihe study involved phosphofructokinase purified from rabbit muscle. Each point in Figure 10-50 represents the catalytic activity of the enzyme that was expressed during incubation in separate test tubes. All of the test tubes... 

The stability constants for ATP and a variety of cations can be used in the following way. One might be concerned that the high levels of potassium ions in the cell could form a K-ATP complex and thus prevent formation of the Mg-ATP complex required by a variety of enzymes. This concern may arise from the fact that the level of K in the cell is about tenfold greater than that of Mg. From Table... 

Magnesium ions bind to inorganic phosphate [P,] and to citrate. The levels of these anions, respectively, are about 3 3 and 1,2 mM in the cytosol and 17.0 and 5-2 mM in the mitochondria. The association constants for Mg-P and Mg-citrate formation are small, compared with that for Mg-ATP formation (see Table 10.16), Pj and citrate might be ex peeled to only slightly impair the formation of the Mg-ATP complex in the cell,... 

Methylcobalamin, 516 5-Melhyl-cytosine, deaminatitm, 894 Methylglyoxal, 836 Methylmalonic acid (MMA), 434, 522 MethylmalcHiy. CoA, 434, 517, 518 Mevalonic acid, 327, 328 Mg-ATP complex, 795-796 Micelles, 25,27-29 MLcroaulophagy, 444 Microbiological assays, 508 biotin determination, 541 folate status by, 509 thiamin status, 607 Microcytic anemia, 5H Microsomal ethanol-oxidizing system, 247 Microvilli, 58 Milk... 

This formula indicates that the concentration of the MP complex is dependent on those of M and P. The formula can be used to estimate the concentration of MP, with a knowledge of the total Mg and total ATP. The cytosolic level of total Mg is about 10 mM and that of ATP is about 2 mM. The value of K is taken from Table 10.16. The concentration [M] of free magnesium is 10 - [MP]. The concentration [P] of free ATP is 2.0 - [MP]. The value for the concentration [MP] of the Mg-ATP complex can easily be foimd from the following formula using a computer ... 

FIGURE 10.4 Nucleophilic attack of the C6—OH group of glucose on the y-phosphate of Mg " "—ATP complex. (Adapted from Voet [Pg.201]

ALA-dehydratase, and isocitrate dehydrogenase and decreases Na+, K -ATPase activity, Mg +-ATPase activity, and choline uptake into synaptosomes. In vitro, aluminum displaces magnesium from Mg +-ATP complexes, and it could thus antagonize virtually any phosphatetransferring reaction that uses Mg +-nucleotide triphosphate complexes. 

Following the preparation of the jS-y complex of Cr +ATP, careful chromatography yields four isomers of the complex, two l and two d. Initial studies were performed with each of the purified diastereomers on hexokinase 61). Analysis demonstrated that only one isomer, A, served as a substrate for this enzyme and that the other diastereomer was unreactive. Subsequent studies on other kinases demonstrated varying selectivity for different kinases for the A or A isomers. These studies were performed with either complexes of ADP or ATP. Selectivity was based on potency of inhibition of the various isomers in many cases and the observation of single-turnover reaction in several other cases. If analogy of these structures with those of Mg ADP and Mg ATP complexes holds, individual enzymes have different stereoselectivities for the M-nucleotide structures. A summary of the selectivity of varying M-nucleotide complexes for some enzymes is presented in Table IV 61-65). 

The reaction mechanism of the mammalian enzyme appears to involve binding of the substrates in an ordered sequence as follows ATP and divalent cation (a Mg +-ATP complex) binds to the enzyme followed by glutamate which reacts to form an enzyme bound y-glutamyl phosphate (this can occur in the absence of ammonia), ammonia then binds to the enzyme, attacks the phosphoryl group resulting in the formation of a tetrahedral intermediate before release of the products. The ammonia-binding site appears to be hydrophobic which would suggest that unionized ammonia and not the ammonium ion is the substrate (Meister, 1974). 

All enzymatic reactions involving ATP require ion as an activator. These types of reactions are very common in nature, especially with kinases. In such cases, the true substrate is Mg ATP complex, that is, a substrate-activator complex, and free ATP molecules are not the active substrates of enzymes. In addition to forming an active complex with substrate, metal ions may also combine with the enzyme at an additional specific activation site, this additional binding site may be essential or nonessential. Thus, the metal ions may be treated as true substrates of enzymes. 

Enzymatic determination The activity of gluco-kinase is dependent on the concentration of the Mg-ATP complex ... 

It has been shown that in solution, the Mg-ATP complex exists in various conformations which are in rapid equilibrium. On the other hand, Cr + and Co + ATP complexes are configurationally stable and isomers (10.105) can be separated by chromatography. The coordination chemistry of ATP is of much current interest [86]. 

K+ and the other a Mg++-ATP complex. When Na+ replaces K+, the system decreases in activity. 

---