Double-displacement Mechanisms

Maltose phosphorylase cannot carry out a similar reaction. The P exchange reaction of sucrose phosphorylase is accounted for by a double-displacement mechanism where E = E-glucose ... 

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. 

For either of the ternary complex mechanisms described above, titration of one substrate at several fixed concentrations of the second substrate yields a pattern of intersecting lines when presented as a double reciprocal plot. Hence, without knowing the mechanism from prior studies, one can not distinguish between the two ternary complex mechanisms presented here on the basis of substrate titrations alone. In contrast, the data for a double-displacement reaction yields a series of parallel lines in the double reciprocal plot (Figure 2.15). Hence it is often easy to distinguish a double-displacement mechanism from a ternary complex mechanism in this way. Also it is often possible to run the first half of the reaction in the absence of the second substrate. Formation of the first product is then evidence in favor of a doubledisplacement mechanism (however, some caution must be exercised here, because other mechanistic explanations for such data can be invoked see Segel, 1975, for more information). For some double-displacement mechanisms the intermediate E-X complex is sufficiently stable to be isolated and identified by chemical and/or mass spectroscopic methods. In these favorable cases the identification of such a covalent E-X intermediate is verification of the reaction mechanism. 

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. 

As one might expect the rate of p-nitrophenyl heptanoate hydrolysis increased at low ethanol concentrations as a result of apolar binding. The rate of p-nitrophenyl acetate hydrolysis also increased markedly at low ethanol concentration. This finding was explained by a conformational effect on the polymer, that is, lower ethanol concentration brings about a shrinkage of the polymer, which increases concerted interactions of the imidazole residues. The hydrolysis of 3-nitro-4-dodecanoyloxybenzoate was found to be 1700 times faster in the presence of poly[4(5)-vinylimidazole] compared to free imidazole (77). A double-displacement mechanism was demonstrated for this system (75). 

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

CONCENTRATION-RESPONSE CURVE Double displacement mechanism,... 

Such behavior is consistent with the double-displacement mechanism of Scheme 5.2, in which fast acylation of the catalyst (cat) [Eq. (4)] is followed by slower deacylation of the acylated form catAc, [Eq. (5)]. 

Analysis ofthe kinetic data shows that the barium salt of 7, as well as the analogous salts of its higher homologues, perform much less efficiently than 4-Ba. The Ba complex of 7 turns over with a very low efficiency, caused by the extreme slowness of the deacylation step. Only a minor fraction ofthe liberated pNPOH in the steady-state phase is due to the expected double displacement mechanism. A larger fraction is most likely ascribable to the metal ion not sequestered by 7, and thereby available in solution for electrophilic assistance to direct methanolysis of the ester reactant. 

The data reported in Tables 5.4 and 5.5 show that the alkaline-earth metal ion complexes are suitable to catalyze with turnover the methanolysis of pNPOAc via the double displacement mechanism of Scheme 5.2. Time-concentration profiles for the... 

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. ... 

Four kinds of experiments were used to identify this double-displacement mechanism. 

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. 

A double-displacement mechanism is unlikely, for D-glucose is not found in a system containing potato phosphorylase, arsenate, and D-glucosyl phosphate. ... 

---