Enzymes utilizing ketimine intermediates

The mechanism for the uncatalyzed decarboxylation of P-ketoacids had previously been established by Bredt and by Pedersen (Bredt, 1927 Pedersen, 1929 1936 Westheimer and Jones, 1941). The acid loses C02 to form the enol of the product, which subsequently ketonizes. The idea behind Pedersen s mechanism for aniline catalysis is that nitrogen is more basic than oxygen, and so could be protonated more readily the protonated imine would provide a better electron sink than the ketone. Although Pedersen offered little or no experimental support for his hypothesis, it provided a basis in physical organic chemistry for the mechanism of the corresponding enzymic process. 

Hamilton marked the carbonyl group of acetoacetic acid with ieO, and then carried out the enzymic decarboxylation (Hamilton and Westheimer, 1959). The product of the decarboxylation, acetone, contained none of the label. This result is demanded by the ketimine mechanism, whereas the mechanism of uncatalyzed decarboxylation would have required that the label appear intact in the product. Of course, in order to make these statements we had to carry out an elaborate set of control experiments, since 180 is washed out of both acetone and acetoacetic acid by buffers and even more 

The general idea of this mechanism was promptly confirmed both for the decarboxylation of acetoacetate, and for the physiologically much more important reaction of transaldolase. In both cases, the development depended upon a new tool introduced in 1958 by Edmond Fischer and 

Krebs (Fischer et al 1958). They discovered that pyridoxal phosphate is attached to phosphorylase A by a ketimine linkage, and that the C=N bond of this linkage could be irreversibly reduced with sodium borohydride. Pyridoxal phosphate does not participate directly in the enzymic reaction of phosphorylase the significance of the work rests on the fact that the reduction occurs without inactivating the enzyme. In 1961, Horecker and his coworkers reduced a mixture of glucose-6-phosphate-14C and transaldolase 

In 1962 too, Fridovich showed that the addition of sodium borohydride to a mixture of acetoacetate decarboxylase and acetoacetate inactivates the enzyme, whereas the addition of borohydride to a buffered solution of the enzyme alone has no effect on the rate at which it can promote the decarboxylation of acetoacetate (Fridovich and Westheimer, 1962) this work confirmed the ketimine mechanism that had previously been advanced for the decarboxylation. Subsequent work (beyond the scope of this review) showed that the reaction product, on hydrolysis, yielded e-isopropyllysine [8], formed by the reduction of the ketimine of acetone (11), and control experiments showed that this ketimine was actually an intermediate in the enzymic pathway, as had been postulated (Warren et al., 1966). 

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A second mode of reaction of the quinonoid-carban-ionic intermediate is utilized by plants which synthesize an enzyme that acts on the amino acid S -adenosylmethionine to form a cyclic three-membered ring compound aminocy-clopropane carboxylic acid. This is a major plant hormone. In a third type of reaction a proton is added back to the coenzyme itself (see Fig. 14) to form what is called a ketimine (not illustrated). This is a Schiff base of pyridox-amine phosphate (PMP, Fig. 5) with an a-oxoacid and is an essential intermediate compound in the important process of transamination (Fig. 14). This process is utilized by all living organisms both in the synthesis of amino acids and in the breakdown of excesses of amino acids. The human body forms several amino acids via transamination. As shown in Fig. 15, this is a reversible sequence involving a cyclic interconversion of PLP and PMP in reaction steps of the type illustrated in Fig. 14. 

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