Enzyme double displacement

Reactions that fit this model are called ping-pong or double-displacement reactions. Two distinctive features of this mechanism are the obligatory formation of a modified enzyme intermediate, E, and the pattern of parallel lines obtained in double-reciprocal plots (Figure 14.19). 

FIGURE 14.22 Glutamate aspartate aminotransferase, an enzyme conforming to a double-displacement bisnbstrate mechanism. Glutamate aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the —NH, acceptor from glntamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine enzyme is the E form.)... 

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. 

Many other multisubstrate examples abound in metabolism. In effect, these situations are managed by realizing that the interaction of the enzyme with its many substrates can be treated as a series of uni- or bisubstrate steps in a multi-step reaction pathway. Thus, the complex mechanism of a multisubstrate reaction is resolved into a sequence of steps, each of which obeys the single- and double-displacement patterns just discussed. 

The antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

Figure 2.15 Double recipcrocal plots for a bi-bi enzyme reactions that conform to (A) a ternary complex mechanism and (B) a double-displacement (ping-pong) 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. 

A balanced hypothetical chemical equation indicating the transfer of electrons between two different oxidation states of the same element of chemical species. 2. One segment of a ping-pong (or double-displacement) enzyme mechanism. 

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

An enzyme-catalyzed double-displacement reaction mechanism in which two substrates react to form two products, but one product must be released before the second substrate binds to the enzyme. 

Km and Umax have different meanings for different enzymes. The limiting rate of an enzyme-catalyzed reaction at saturation is described by the constant kcat, the turnover number. The ratio kcat/Km provides a good measure of catalytic efficiency. The Michaelis-Menten equation is also applicable to bisubstrate reactions, which occur by ternary-complex or Ping-Pong (double-displacement) pathways. 

Although this reaction is fully reversible, the relatively high [ATP]/[ADP] ratio in cells normally drives the reaction to the right, with the net formation of NTPs and dNTPs. The enzyme actually catalyzes a two-step phosphoryl transfer, which is a classic case of a double-displacement (Ping-Pong) mechanism (Fig. 13-12 see also Fig. 6-13b). First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme... 

Two possible explanations for the lack of inversion during this reaction are that the enzyme acts by a double-displacement reaction or through a stabilized carbocationic intermediate. Let us consider these possibilities in turn. 

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... 

A large number of possible enzyme-substrate complexes may form, e.g. the binary complexes EA, EB, EP and EQ, ternary complexes EAB, EPQ, EAQ, and EBQ. Most two-substrate reactions can be grouped into two major classes, based upon the reaction sequence in the two-substrate reactions, single displacement reactions and double displacement reactions. 

In bi-substrate reactions of the double displacement type, one substrate must be bound and one product released before the entry of the second substrate. In such reactions, the first substrate reacts with the enzyme to yield a chemically modified form of the enzyme (usually a functional group is changed) and the first product. In the second step, the functional group of the modified enzyme is transferred from the enzyme to the second substrate to form the second product. A good example is the aminotransferase class of enzymes, where an amino group is transferred from an amino-acid to the enzyme, from which it is transfered to a keto-acid. 

Enzymes often require multiple substrates to complete their catalytic cycle. This may involve combining two compounds into one molecule or transferring atoms or electrons from one substrate to another. The substrates may both bind to an enzyme and react collectively, or each substrate might bind, react, and release sequentially. With two substrates, if both bind to the enzyme, a ternary complex (ES S2) will form (Scheme 4.8). The order of substrate addition may be important (ordered) or not (random order). Cases in which the two substrates react sequentially follow a double-displacement, or ping-pong, mechanism (Scheme 4.9). Enzymes requiring more than two substrates have more complicated complexation pathways. 

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). 

---