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

Types of enzyme-catalyzed reactions
Nucleophilic substitution reactions (Type 1 reactions)
Mechanism of double displacement reactions

It is reasonable to assume that sucrose phosphorylase catalyzes two consecutive substitution steps, each proceeding with inversion [Eq. (7-6)].

In this case, The First stage of reaction (7-6) would involve the Displacement of the enzyme's nucleophilic group (step a) to form a glycosyl-enzyme intermediate, while the Second Stage (step b) would involve a phosphate attack, resulting in the regeneration of the enzyme and the free nucleophilic group B^-.

What are the possible predictions and experimental tests to prove a double-displacement mechanism? Section B, 3, a discusses four types of experiments performed with sucrose phosphorylase, which can also be applied to The Study of many Other Enzymes.

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a. Exchange reactions

Based on the double-displacement mechanism, one would predict that the enzyme should catalyze partial exchange reactions between one of the two substrates and a labeled product. For instance, sucrose containing ¹⁴C in its fructose moiety should react with sucrose phosphorylase to yield a glucosyl-enzyme and free radioactive fructose as a substitution product:

Here, asterisks denote compounds containing ¹⁴C. Typically, very low molar enzyme concentrations are used in such experiments; therefore, to detect reaction (7-7), it is sufficient to add a large excess of non-radioactive fructose. Under these conditions, the enzyme does not catalyze the overall chemical reaction, but instead repeatedly cycles between the free enzyme and glucosyl-enzyme states. In each reverse reaction, the enzyme preferentially utilizes unlabeled fructose. The net result is that the enzyme catalyzes an exchange reaction between fructose and sucrose:

The exchange between fructose and sucrose has been observed experimentally [7], as has the second predicted exchange—namely, the exchange of glucose-1-phosphate with radioactive phosphate:

Closely related to these exchange reactions are transfer reactions, in which a ketose other than fructose, such as D-ketoxylose, reacts with the glucosyl-enzyme to form a new disaccharide containing glucose and the added ketose [7].

The analysis of exchange reactions as a criterion for mechanism is widely used in studying complex enzymatic processes across all areas of METABOLISM and therefore requires a thorough understanding. At the same time, one must be aware of the potential limitations of this criterion. While the double-displacement mechanism predicts the occurrence of specific exchange reactions, the experimental detection of such processes does not strictly prove the existence of covalently linked enzyme intermediates. Furthermore, enzymes operating via a double-displacement mechanism do not always catalyze the expected exchange reactions (Section B, 5).

b. Arsenolysis

Sucrose phosphorylase also catalyzes the Cleavage of sucrose by arsenate and promotes the rapid arsenolysis of glucose-1-phosphate to free glucose. This reaction is most easily conceptualized as proceeding via The formation of a glucosyl-enzyme intermediate, yielding an unstable product, glucose-1-arsenate (Addendum 7-A). Arsenolysis serves as a general method for detecting reactive enzyme-bound intermediates that normally react with phosphate groups. Arsenate is one of many substrate analogs that can also be used to channel reactive enzyme-bound intermediates into alternative metabolic pathways.

c. Kinetics

The double-displacement mechanism requires the enzyme to function in a shuttle-like manner, cycling between the free enzyme and a substrate-bearing intermediate, i.e., the glycosyl-enzyme. Kinetic studies of sucrose phosphorylase-catalyzed reactions demonstrate that the dependence of reaction velocity on the concentrations of sucrose and HPO^2-₄ conforms to the laws governing a ping-pong mechanism (Chapter 6, Section A, 10, a) [8].

a. Direct isolation of intermediates

Methods for isolating pure enzymes and handling extremely small quantities of substances are now so advanced that it is frequently possible to directly confirm the existence of postulated enzyme-bound intermediates. For example, the glucosyl-enzyme has been successfully isolated in a denatured form following the reaction between highly purified sucrose phosphorylase and isotopically labeled sucrose [8]. The glucosyl-enzyme is too labile to withstand even mild peptide backbone cleavage and the Isolation of the glycosyl-labeled fragment. However, indirect evidence strongly indicates that —B: is a carboxylate group (—COO-) [9].