The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5′-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

Acute pancreatitis (AP) is characterised by self-digestion of the pancreas by its own proteases. This pathophysiological initiating event in AP occurs inside pancreatic acinar cells where intrapancreatic trypsinogen becomes prematurely activated by cathepsin B (CTSB), and induces the digestive protease cascade, while cathepsin L (CTSL) degrades trypsin and trypsinogen and therefore prevents the development of AP. These proteases are located in the secretory compartment of acinar cells together with cystatin C (CST3), an endogenous inhibitor of CTSB and CTSL. The results are based on detailed biochemical analysis, site-directed mutagenesis and molecular dynamics simulations in combination with an experimental disease model of AP using CST3 deficient mice. This identifies that CST3 is a critical regulator of CTSB and CTSL activity during AP. CST3 deficient mice show a higher intracellular CTSB activity resulting in elevated trypsinogen activation accompanied by an increased disease severity. This reveals that CST3 can be cleaved by trypsin disabling the inhibition of CTSB, but not of CTSL. Furthermore, dimerised CST3 enhances the CTSB activity by binding to an allosteric pocket specific to the CTSB structure. CST3 shifts from an inhibitor to an activator of CTSB and therefore fuels the intrapancreatic protease cascade during the onset of AP.