beta-Alanine synthase: purification and allosteric properties.
beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.
Matthews
M M
MM
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599-7260.
Liao
W
W
Kvalnes-Krick
K L
KL
Traut
T W
TW
D000495:Allosteric Site; D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006358:Hot Temperature; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000581:Amidohydrolases; C021590:beta-ureidopropionase