Enzymes double-displacement mechanism

Maltose phosphorylase proceeds via a single-displacement reaction that necessarily requires the formation of a ternary maltose E Pi (or glucose E glucose-l-phosphate) complex for any reaction to occur. Exchange reactions are a characteristic of enzymes that obey double-displacement mechanisms at some point in their catalysis. 

Kirsh et al. 42) prepared apolar derivatives of poly(4-vinylpyridine) by benzylation. With nitrophenyl acetate as the substrate the benzylated catalyst is 100 times more effective than 4-ethylpyridine. A double-displacement mechanism was observed. The rate constants for deacylation of the acylpoly(vinylpyridine) derivatives were about 4 x 10" /sec. The comparable value for a-chymotrypsin is 8 x 10 /sec. The factor of 20 seems small, but it should be kept in mind that deacetylation of a-chymotrypsin is very slow compared with the deacylation reactions involving the natural substrates of the enzyme. 

PING PONG HALF-REACTIONS. Many enzymes operate by double-displacement mechanisms involving covalent enzyme-substrate intermediates as shown in the following scheme ... 

Double-displacement mechanisms. In a doubledisplacement mechanism sucrose phosphorylase would catalyze two consecutive single displacements, each with inversion. A nucleophilic group of the enzyme would react in Eq. 12-7, step a. In step b, a phosphate would react to regenerate the enzyme with its free nucleophilic group -B. ... 

Kinetics. In a double-displacement mechanism the enzyme shuttles between free enzyme and the intermediate carrying the substrate fragment (here, the glycosyl enzyme). With sucrose phosphorylase the maximum velocity varies with the concentrations of sucrose and HP042 in the characteristic fashion expected for this "ping-pong" mechanism (Eq. 9-47)43... 

Exchange reactions. In a double-displacement mechanism sucrose containing 14C in the fructose portion of the molecule should react with free enzyme E to form glycosyl enzyme and free radioactive fructose (Eq. 12-8). The 14C-containing groups are designated here by the asterisks. 

This acetyl enzyme hydrolyzes very slowly at pH 4 but rapidly at higher pH. These experiments suggested a double displacement mechanism ... 

The pepsin family is most active in the low pH range 1-5. All of the enzymes contain two especially reactive aspartate carboxyl groups.378 One of them (Asp 215 in pepsin) reacts with site-directed diazoni-um compounds and the other (Asp 32) with site-directed epoxides.379 It is attractive to think that one of these carboxyl groups might be the nucleophile in a double displacement mechanism. The second carboxyl could then be the proton donor to the cleaving group. 

The alkaline phosphatase of E. coli is a dimer of 449-residue subunits which requires Zn2+, is allo-sterically activated by Mg2+, and has a pH optimum above 8.667/708 711 At a pH of 4, incubation of the enzyme with inorganic phosphate leads to formation of a phosphoenzyme. Using 32P-labeled phosphate, it was established that the phosphate becomes attached in ester linkages to serine 102. The same active site sequence Asp-Ser-Ala is found in mammalian alkaline phosphatases. These results, as well as the stereochemical arguments given in Section 2, suggest a double-displacement mechanism of Eq. 12-38 ... 

The ping-pong (or substituted-enzyme or double-displacement) mechanism... 

Substrate specificity differences between boar acrosin and trypsin are not particularly manifest when using small substrates, but these enzymes show distinctly different kinetics of porcine ZP hydrolysis (34). The loss of 30% mass in the conversion from m - to m -acrosin has little effect on the kinetic analyses of inhibition and substrate preference with artificial substrates and small trypsin inhibitors, indicating that this excised portion of the enzyme contributes little to the topography of the active site (35). From Km analyses with amide and ester substrates of Arg and Lys, acrBSin prefers the Arg substrates over Lys, and Km differences between amide and ester substrates indicates that ac Ssin proceeds kinetically through a classical double displacement mechanism as does trypsin (36). 

Two specific examples from Table 2 are worthy of further mention. Nucleoside phosphotransferase, which catalyzes Equation 20, had been proposed not to involve a double-displacement mechanism or an intermediate phosphoryl-enzyme. 

In double-displacement, or Ping-Pong, reactions, one or more products are released before all substrates bind the enzyme. The defining feature of double-displacement reactions is the existence of a substituted enzyme intermediate, in which the enzyme is temporarily modified. Reactions that shuttle amino groups between amino acids and a-keto acids are classic examples of double-displacement mechanisms. The enzyme aspartate aminotransferase (Section 23.3.1) catalyzes the transfer of an amino group from aspartate to a-ketoglutarate. 

Many clinically important yff-lactamases are serine proteases that catalyse y5-lactam hydrolysis by a double displacement mechanism involving a covalent acyl-enzyme intermediate. Inhibitors of these enzymes exert their effect by the formation of a stable acyl-enzyme complex. In most cases, this is as a result of changes that take place in the acyl residue after interaction with the enzyme, that is, the inhibitors are mechanism-based. In other cases, the inhibition of yS-lactamases may merely be due to the formation of a relatively stable covalent acyl-enzyme complex without additional alteration [31]. 

Rhodanese is present in the mitochondria, particularly of liver and kidney cells. A double-displacement mechanism has been proposed for its biochemical action. The steps are as follows The free enzyme reacts with a sulfane sulfur-containing compound (a sulfane sulfur is one that is divalent and covalently bonded only to other sulfurs), cleaving the S-S bond of the donor substrate (e.g., SSOj ) to form the sulfur-substituted enzyme. The latter reacts with the cyanide (a thiophilic acceptor) to form thiocyanate in an essentially irreversible reaction. 

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