Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of enzyme-catalyzed reactions
Substitution at the phosphorus atom
Nuclear relaxation induced by paramagnetic ions

The Use of paramagnetic ions (e.g., Mn²⁺, Cu²⁺, or Cr³⁺) to induce nuclear relaxation in substrate and coenzyme molecules located within enzyme active sites is a highly effective research method, and its scope is continually expanding [79–83]. Flavin radicals and selectively introduced nitroxide spin labels can also serve this purpose. It is well established that paramagnetic ions affect the magnetic relaxation of neighboring nuclei (see Appendix 5-A). For example, trace amounts of Mn²⁺ in a sample cause line broadening corresponding to ¹H, ¹³C, and ³¹P in conventional NMR spectra.

Valuable information about Enzymes can sometimes be obtained from the Analysis of the NMR signal of protons in Water (the solvent). The relaxation time of free water protons is typically greater than 1 s. However, the protons of coordinated water molecules in the Mn(H₂O)₆²⁺ ion exhibit much higher relaxation rates (T₁ and T₂ values on the order of 10^-5 s). Since coordinated water molecules generally exchange very rapidly with molecules in the surrounding medium, a small amount of manganese ions can substantially increase the relaxation rate of protons across all water molecules. Using appropriate Methods, one can observe proton line broadening in the NMR spectrum along with changes in T₁ and T₂ values. It is known that the magnitude of a paramagnetic ion's effect on the magnetic relaxation of a neighboring Nucleus is inversely proportional to the sixth power of the internuclear distance. Assuming that the distance between Mn²⁺ and H in the hydrated Mn²⁺ ion is 0.287 ± 0.005 nm, one can establish quantitative relationships between, on the one hand, the changes in T₁ and T₂ values and, on the other hand, the number of water molecules in the coordination sphere of the protein-bound metal ion at any fixed moment in time, as well as their exchange rate with solvent molecules.

Although recording Changes in the magnetic relaxation of ¹H, ¹³C, and ³¹P nuclei encounters even greater difficulties, such measurements can yield exceptionally useful insights into active-site geometry. These calculations rely on the principle that the effect on nuclear relaxation decreases with the internuclear distance r in proportion to 1/r⁶. Although The Theory of these phenomena is quite complex, under certain conditions the relevant equations simplify to the following form:

Class="center">r = C[T_1Mf(τ_c)]^1/6, (7-24)

where r is the internuclear distance; C is a constant representing a specific combination of physical constants; T_1M is the longitudinal relaxation time induced by the paramagnetic ion; and f(τ_c) is a complex function dependent on the correlation time τ_c (observed at the nuclear Resonance frequency) and the precessional frequency of electron spins in the paramagnetic centers. A variety of methods exist for estimating τ_c and r using equation (7-24).

FIG. 7-6. Structure OF THE creatine kinase—Mn²⁺—ADP—creatine phosphate complex (after McLaughlin et al. [84]). The arrangement of substrates is shown in accordance with distances measured by magnetic resonance methods.

An example of such studies [84] is presented in Fig. 7-6. The NMR spectra of the bound Mn²⁺ ion and the nitroxide spin label were measured using a 220 MHz pulse spectrometer. In addition, EPR spectral measurements made it possible to determine the distance between the Mn²⁺ ion and the nitroxide radical. The manganese ion is coordinated to the α- and β-phosphates of ADP in a manner similar to that illustrated in scheme (7-23).

ADP and ATP complexes with Cr³⁺ were also prepared and tested as substrates in various enzymatic reactions. It turned out that Kinases are completely inactive toward these complexes, which may be due to improper or excessively tight binding of the Cr³⁺ ion. Apparently, during the catalytic process, the ion or its surrounding groups undergo a positional shift over a certain distance, whereas in Cr³⁺ complexes this movement is restricted.

Appendix 7-D

Magnesium

An average adult human ingests 10–12 mmol of magnesium ions daily (∼¹/₄ g). Of this amount, approximately one-third is absorbed from the digestive tract. To maintain Homeostasis, an equivalent amount of magnesium is excreted in the urine. Sixty percent of the body's magnesium resides in bones. The concentration of serum Mg²⁺ is ∼0.85 mM, whereas the total magnesium concentration in Tissues ranges from 5 to 8 mM. However, only ∼1 mM of Mg²⁺ exists in the body in a free state. The remainder is bound to Proteins and soluble compounds such as ATP, ADP, and other phosphate- and carboxylate-containing molecules.

It has been suggested that the concentration of Mg²⁺ ions, much like that of H⁺ ions, remains in dynamic equilibrium with Blood serum³. Nevertheless, situations likely occur in which at least temporary shifts take place in the concentrations of free Mg²⁺ and free H⁺ ions^б. During rapid Carbohydrate Catabolism, Glycolysis can lead to the acidification of Muscle Cells, causing the pH to drop from 7.3 to 6.3. This pH decrease brings about a marked reduction in the extent of Mg²⁺ binding to molecules such as ATP and a temporary surge in free Mg²⁺ concentration. Similarly, the release of diphosphoglycerate from its complex with Hemoglobin upon oxygenation leads to a drop in free Mg²⁺ concentration, as the ion binds to diphosphoglycerate^в. These fluctuations in free Mg²⁺ concentration may be of great significance in Metabolic Regulation^г.

Magnesium ions possess a smaller ionic radius compared to Ca²⁺ ions, which accounts for their greater ease of penetration into cells. The Role of Mg²⁺ can frequently be assumed by Mn²⁺ with full retention of activity in enzymes that require Mg²⁺ ions. Conversely, high concentrations of Ca²⁺ often antagonize Mg²⁺. This antagonism^д is clearly manifested when studying the combined effects of these ions on cellular excitability. Magnesium deficiency or calcium excess in the environment leads to increased excitability; conversely, an excess of magnesium results in its loss. Interestingly, hibernation in animals is characterized by elevated levels of Mg²⁺ ions.

The functioning of many enzymes involves Mg²⁺, with phosphotransferases (Chap. 3, Sec. B,5) constituting the largest independent group, for which MgATP can be regarded as the substrate. Mg²⁺-dependent enzymes also include Phosphatases and Other Enzymes catalyzing Phosphate group transfer. A specialized function of magnesium relates to its participation in Photosynthesis as a component of chlorophyll.

One of the most toxic metals is beryllium. It has been found that for numerous enzymes, including phosphoglucomutases and phosphatases, Be²⁺ competes with Mg²⁺ for binding at specific sites.

^а Veloso D., Guynn R. W., Oskarrson M., Veech R. L. (1973). J. Biol. Chem., 248, 4811—4819.

^б Purich D. L., Fromm H. J. (1972). Curr. Top. Cell. Regul., 6, 131—167.

^в Bunn H. F., Ransil B. J., Chao A. (1971). J. Biol. Chem., 246, 5273—5279.

^г Rubin H. (1975). Proc. Nat. Acad. Sсi. USA, 72, 3551—3555.

^д Meli J., Bygrave F. L. (1972). Biochem. J., 128, 415—420.