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

Types of reactions catalyzed by enzymes
Substitution at the phosphorus atom
Kinases

A large Class of Enzymes catalyzes the Transfer of phosphate groups from one atom to another¹⁾. These enzymes include kinases, i.e., enzymes that transfer phosphate groups from polyphosphates, such as ATP, to oxygen, nitrogen, or sulfur atoms of a second substrate. An example of a kinase is hexokinase [equation (6-91); Fig. 9-7], the enzyme responsible for the Synthesis of glucose-6-phosphate from free glucose [equation (7-23)].

Hexokinase, like most enzymes catalyzing The transfer of phosphate groups, exhibits an absolute requirement for a divalent cation, typically the Mg²⁺ ion. Although the true substrate for hexokinase is considered to be the ATP–Mg²⁺ complex, the exact mechanism of metal-polyphosphate complex formation on the enzyme surface remains unknown. Presumably, the metal binds to groups of both the enzyme and the substrate.

The scheme shown in equation (7-23) is hypothetical and illustrates a possible function of the metal ion in these reactions. By interacting with the two phosphate groups of the ATP molecule, which become leaving groups in the substitution reaction, the metal ion attracts electrons and facilitates bond Cleavage. According to an alternative mechanism, the metal in the ES complex is located near the terminal phosphate group (the one undergoing transfer), where it may serve to neutralize negative charge and facilitate the approach of the attacking nucleophile. However, if the metal forms a bridging bond between the First and Second phosphates, a stable chelate ring is formed, which would hinder rather than facilitate the reaction. Thus, THE POSITION OF the metal shown in equation (7-23) should be regarded simply as the most probable one. It is also possible that in Ribonuclease, certain basic groups of the enzyme neutralize the negative charge on the terminal phosphate.

¹⁾ Phosphatases are also phosphotransferases that transfer phosphate residues to hydroxyl ions of the aqueous medium. However, such enzymes are usually classified as Hydrolases rather than transferases.

Equation (7-23) illustrates a linear type of attack by the hydroxyl group of glucose. This mechanism of direct, concerted substitution seems quite likely, as no partial exchange reactions have been detected. Furthermore, kinetic studies of the reaction have provided no evidence for a ping-pong mechanism that would indicate The intermediate formation of a phosphoenzyme. Nevertheless, this possibility cannot be ruled out [74]. It is conceivable that both substrates must bind to the Active Site before the enzyme attains the conformation required for catalysis. A phosphorylated enzyme intermediate might possess exceptionally high reactivity, making its detection difficult. For certain phosphotransferases, relatively stable phosphoenzymes have been obtained by Treatment with γ-³²P-ATP in the absence of a second substrate [75]. Mildvan [67] suggested that these phosphoenzymes are formed As a result of Side Reactions indicative of the existence of metaphosphate intermediates.

The three-dimensional structures of Yeast hexokinase (Fig. 7-5, A) [76], phosphoglycerate kinase consisting of 355 amino acid residues (Fig. 7-5, B) [77], and adenylate kinase (Supplement 3-A) [78] have been described recently. The latter enzyme, with a Molecular Weight of ~ 22,000, is the smallest known kinase. In all three cases, the protein molecule consists of two lobes (or domains). For phosphoglycerate kinase and adenylate kinase, the Location OF THE ATP-binding site has been determined. It was found that the two phosphate groups closest to adenosine, P_a and P_β, bind to the same domain (lower domain A in Fig. 7-5, B), whereas the terminal phosphate group of ATP (P_γ) projects into the cleft between the two domains. The Mg²⁺ ion binds to P_a and P_β as shown in scheme (7-23).

FIG. 7-5. A. Schematic representation of the polypeptide chain of a yeast hexokinase subunit. The glucose-binding site is designated by the letter G, and the AMP-binding site by the letter A. The ATP-binding sites in the region between two subunits in various crystal forms are designated by Iu and Id (after Flotow et al. [76]). B. Structure OF THE phosphoglycerate kinase molecule. Cylinders representing α-helical segments of the chain are numbered with Roman numerals starting from the N-terminus. Arrows represent individual strands in the β-structure and their direction. The arrows are labeled alphabetically starting from the N-terminus. The ATP-binding domain is located at the bottom of the figure (after Blake and Evans [77]).

Protein kinases (Chap. 6, Sec. E,2) are currently of great interest; they catalyze the transfer of phosphate groups from ATP to the hydroxyl groups of specific Serine or Threonine residues in their substrate molecules. A characteristic feature of phosphorylation sites in substrate Proteins is the presence of a Lysine or Arginine residue separated from the serine or threonine by only a single amino acid residue [78a].

Due to its strong inhibitory effect resulting from tight binding, and several other properties, the planar nitrate ion can be considered an analog of the transferred phosphate group in the trigonal bipyramidal transition states [equation (7-20)] for kinase-catalyzed reactions [78b]. Another ion of interest as a highly reactive analog of the phosphate ion is the ferrate ion, FeO^2-₄. Although a strong oxidizing agent, the ferrate ion is structurally and acid-base comparable to the phosphate ion and can be used to modify phosphate-binding sites [78c].