On the mechanism of action of yeast endo-polygalacturonase on oligogalacturonides and their reduced and oxidized derivatives.

Yeast polygalacturonase catalyzes a random hydrolysis of pectic acid to a mixture of digalacturonic and galacturonic acids via a series of oligogalacturonides (1). This enzyme will be termed yeast endo-polygalacturonase to differentiate it from end group-hydrolyzing enzymes, which have been named exopolygalacturonases (2). Evidence has been presented that yeast endo-polygalacturonase is a single enzyme (3). Pentagalacturonic acid undergoes a (2 + 3) and a (4 + 1) cleavage during hydrolysis, but tetragalacturonic acid undergoes cleavage into trigalacturonic and galacturonic acids only. The remainder of the hydrolysis consists of a very slow breakdown of the trimer into dimer and monomer. The rate of hydrolysis of tetragalacturonic acid is much faster than that of the trimer, but it is appreciably slower than the rate of breakdown of pectic acid. In order to explain the decreasing rate of hydrolysis with a shortening of the oligouronide chain length, Demain and Phaff (1) postulated that one of the end groups in an oligoor polyuronide chain hinders hydrolysis when the linkage in question is close to this end group. This view would also explain the inability of yeast endo-polygalacturonase to attack digalacturonic acid. Support for this hypothesis would be obtained if it could be shown that the hydrolysis of tetragalacturonic acid occurs at only one end of the chain. Accordingly, a series of oxidized and reduced derivatives of the oligouronides was prepared and used as substrates. Thus if terminal hydrolysis is still possible, cleavage at the bond farthest from the modified reducing group would produce galacturonic acid from the tetramer. If cleavage occurs at the bond adjacent to the modified reducing group, trigalacturonic acid and r.-galactonic acid or mucic acid would be formed. The results presented below give evidence that the (3 + 1) cleavage of tetragalacturonic acid occurs at the latter bond, and that the bond at the nonreducing end of the chain is naturally protected from attack by yeast endo-polygalacturonase.