Effects of Salts on the Conformation and Catalytic Properties of D-Amino Acid Aminotransferase

Abstract

The effects of salts on the biochemical properties of Damino acid aminotransferase from Bacillus sp. YM-1 have been studied to elucidate both the inhibitory effects of salts on the activity and the protective effects of salts on the substrate-induced inactivation. The results from UVvisible spectroscopy studies on the reaction of the enzyme with D-serine revealed that salt significantly reduced the rate of the formation of the quinonoid intermediate and its accumulation. The kinetic and spectroscopy studies of the reaction with α-[ 2 H]-DL-serine in different concentrations of NaCl provided evidence that the rate-limiting step was changed from the deprotonation of the external aldimine to another step(s), presumably to the hydrolysis of the ketimine. Gel filtration chromatography data in the presence of NaCl showed that the enzyme volume was reduced sharply with the increasing NaCl concentration, up to 100 mM. An additional increase of the NaCl concentration did not affect the elution volume, which suggests that the enzyme has a limited number of saltbinding groups. These results provide detailed mechanistic evidence for the way salts inhibit the catalytic activity of Damino acid aminotransferase.

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis

“…KCl likely inhibited enzymatic activity in vitro by impeding formation of the quinonoid intermediate, a phenomenon observed in other PLP-dependent enzymes. 49,50 Therefore, KCl was removed as a SbnA buffer component in all future experiments. Steady-state kinetics was used to investigate the mechanism of SbnA catalysis.…”

“…Optimal wild-type enzyme activity as monitored by phosphate release was observed with no KCl (0–500 mM tested) at pH 8.0 (6.5–8.5 tested) in a 50 mM Tris buffer (Figure S4). KCl likely inhibited enzymatic activity in vitro by impeding formation of the quinonoid intermediate, a phenomenon observed in other PLP-dependent enzymes. , Therefore, KCl was removed as a SbnA buffer component in all future experiments. Steady-state kinetics was used to investigate the mechanism of SbnA catalysis.…”

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

“…42 In contrast, CysK, which is an O -acetylserine sulfhydrylase and belongs to the type II fold PLP-dependent enzyme family, maintains high activity at high salt concentrations. 43 This is consistent with the CysK structure from Haemophilus influenzae (PDB entry 4HO1) in which no salt bridge with the phosphate of PLP is observed as the Arg of the CGS-like enzymes is replaced with T178 (Figure 4D).…”

“…Beyond examples of type I PLP-dependent proteins, d -amino acid aminotransferase (DAAT, PDB entry 4DAA), a type IV fold PLP-dependent enzyme, shows the formation of a salt bridge between PLP and R50 in the active site (Figure C) and shows ionic strength inhibition . In contrast, CysK, which is an O -acetylserine sulfhydrylase and belongs to the type II fold PLP-dependent enzyme family, maintains high activity at high salt concentrations .…”

“…Beyond examples of type I PLP-dependent proteins, D-amino acid aminotransferase (DAAT, PDB entry 4DAA), a type IV fold PLP-dependent enzyme, shows the formation of a salt bridge between PLP and R50 in the active site (Figure 4C) and shows ionic strength inhibition. 42 In contrast, CysK, which is an Oacetylserine sulfhydrylase and belongs to the type II fold PLPdependent enzyme family, maintains high activity at high salt concentrations. 43 This is consistent with the CysK structure from Haemophilus inf luenzae (PDB entry 4HO1) in which no salt bridge with the phosphate of PLP is observed as the Arg of the CGS-like enzymes is replaced with T178 (Figure 4D).…”