Identification of key amino acid of HbACS2 in rubber tree
[Objective]Acetyl-CoA synthetase(ACS,EC 6.2.1.1)is an important enzyme in cellular metabolism,which catalyzes the formation of acetyl-CoA from acetate and coenzyme A under ATP-dependent conditions.Previous studies found that one member of the ACS family in rubber trees,HbACS2,influenced the metabolic balance of acetate in the laticiferous cells and the capability of latex regeneration.However,what regulates the activity of this enzyme remains unclear.Studies in microorganisms and model plants have shown that ACS enzyme activity is related to the acetylation modification status of its key amino acid sites and some key lysine acid sites.Therefore,in order to understand the regulation mechanism of HbACS2 enzyme activity,it is necessary to screen its key amino acids.[Method]Based on the amino acid characteristics of HbACS2,three conserved lysine(K)residues(K298,K342,and K723)were chosen to be substituted to construct prokaryotic expression vectors via site-directed mutagenesis.They were substituted with alanine(A),arginine(R),and glutamine(Q),respectively.The corresponding variant proteins and the wild-type protein were obtained by induction,expressed and purified according to instructions of Ni-NTA agarose.Using the purified wild-type protein as a control,the differences in substrate acetic acid binding ability and enzyme activity between wild-type and mutant proteins were compared by in vitro enzyme activity analysis.[Result]The findings indicated that the K723 site mutation of HbACS2 had the most significant effect on the enzyme activity.Both the K723A and K723R mutant variants completely lost their enzyme activity,while the K723Q variant exhibited a 180-fold reduction in activity.Furthermore,mutations of K298 significantly reduced their enzyme activities and altered the Michaelis constants for acetate.Specifically,the K298Q variant showed the most drastic alteration,with a 17.85-fold decrease in enzyme activity and a 6.5-fold increase in substrate affinity(Km=0.06 mmol/L).The enzyme activity of K298A decreased by approximately 4-fold,along with nearly a twofold decrease in substrate affinity(Km=0.754 3 mmol/L),whereas K298R exhibited a 3.7-fold decrease in enzyme activity and a twofold increase in substrate affinity(Km=0.182 7 mmol/L).The mutation of K342 exerted the least impact on enzyme activity among the three variant proteins.Specifically,the enzyme activity of the K342R variant decreased by 2.75-fold,with a 5.3-fold increase in acetate affinity(Km=0.074 mmol/L).The enzyme activities of K342A and K342Q decreased by 42%and 14%,respectively,with a 34%and 17%increase in acetate affinity.[Conclusion]These results confirmed the substantial influence of key lysine within HbACS2 on its enzyme activity and substrate affinity,thus providing an important reference for further revealing the regulation mechanism of ACS activity.
万方数据
维普
