title: The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin.
abstract: The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca(2+), are proposed to be important for ISP activity through structural changes. The presence of >0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca(2+) at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions.
authors: School of Biosciences, Cardiff University, Cardiff CF10 3AT, UK.
authors: Michael
authors: M
authors: Gamble
authors: Georg
authors: G
authors: Künze
authors: Andrea
authors: A
authors: Brancale
authors: Keith S
authors: KS
authors: Wilson
authors: D Dafydd
authors: DD
authors: Jones
keywords: Binding simulations; ESP, extracellular subtilisin; ISP, intracellular subtilisin (processed and active); Metal binding; Protease; SEC, size exclusion chromatography; Substrate specificity; Subtilisin; Suc, succinyl; pNA, para-nitroanilide; proISP, unprocessed pro-form of ISP; proISPS250A, proISP with the active site S250A mutation
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