An investigation into the kinetics and regulatory properties of the
type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus
thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and
activated by ADP by modifying the affinity exhibited for the substrate fructose
6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However,
TtPFK binds both of these allosteric ligands significantly more tightly than do
other bacterial PFKs while effecting a substantially more modest extent of
inhibition or activation at 25°C, reinforcing the principle that binding
affinity and effectiveness can be both independent and uncorrelated to one
another. These properties have allowed us to rigorously establish that PEP only
inhibits by antagonizing the binding of Fru-6-P, and not by influencing turnover
– a conclusion that requires kcat be determined under
conditions in which both inhibitor and substrate are saturating simultaneously.
In addition, the temperature dependence of the allosteric effects on Fru-6-P
binding indicate that the coupling free energies are entropy-dominated, as
observed previously for PFK from B. stearothermophilus but not
for PFK from E. coli, supporting the hypothesis that
entropy-dominated allosteric effects may be a characteristic of enzymes derived
from thermostable organisms. For such enzymes, the root cause of the allosteric
effect may not be easily discerned from static structural information such as
might be obtained from X-ray crystallography.
Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.