Subdomain II of {alpha}-Isopropylmalate Synthase is Essential for Activity: Inferring a Mechanism of Feedback Inhibition [Protein Structure and Folding]

August 15th, 2014 by Zhang, Z., Wu, J., Lin, W., Wang, J., Yan, H., Zhao, W., Ma, J., Ding, J., Zhang, P., Zhao, G.-P.

The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product L-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2) which exhibits a catalytic activity comparable to that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 lost about 50% of the catalytic activity; however, complete missing of the regulatory domain up to R385, almost equivalent to the intact LbIPMS2, maintained about 90% of the activity. Moreover, in either LbIPMS2 or LbIPMS1, further deletion of several residues from the C-terminus of subdomain II significantly impaired or completely abolished the catalytic activity. These results define a complete and independently functional catalytic module of IPMS consisting of both the N terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS reveals two different conformations of subdomain II, which likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data lead us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II which might associate with alteration of the binding affinity towards acetyl-CoA.
  • Posted in Journal of Biological Chemistry, Publications
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