Specific, limited tryptic modification of wheat-germ fructose-bisphosphate aldolase subunits: destruction of catalytic activity but not of ability to establish precise subunit-subunit recognition

Abstract

We have been using the glycolytic enzyme fructose-bisphosphate aldolase ( d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) as a model system to investigate the assembly of oligometric enzymes. In the present work we investigate the effect of specific, limited tryptic modification on the properties of aldolase isolated from what germ. The wheat-germ enzyme was selected, since several aldolases isolated from animal sources were not readily susceptible to the specific tryptic modification seen with this plant enzyme. We will show that: (1) Low levels of trypsin cause a first-order inactivation of wheat-germ aldolase activity which is associated with a fairly specific cleavage of the enzyme which reduces its subunit molecular weight from 41 000 to 39 000. (2) The proteolytic modification is greatly inhibited in the presence of the ladolase substrate, fructose biphosphate. (3) The intact and modified enzymes appear to have similar surface changes, as judged by their behavior during electrophoresis in polyacrylamide gels under non-denaturing conditions. (4) The modified aldolase is not specifically eluted from phosphocellulose columns by fructose bisphosphate under the conditions used in the affinity chromatographic isolation of the intact enzyme, suggesting that the modified enzyme may no longer be able to bind substrate. (5) Although enzymatically inactive, the modified aldolase subunits are able to refold and reassociate into tetrameric combinations following unfolding of the subunits by treatment at law pH; thus, this specific proteolytic modification does not interfere with the ability of wheat-germ aldolase subunits to refold and to establish precise subunit-subunit recognition in vitro.