Control of L-ornithine specificity in Escherichia coli ornithine transcarbamoylase. Site-directed mutagenic and pH studies.
Escherichia coli ornithine transcarbamoylase displays a strict specificity toward its second substrate L-ornithine. After forming a binary complex with carbamoyl phosphate and undergoing an induced-fit isomerization (Miller, A. W., and Kuo, L. C. (1990) J. Biol. Chem. 265, 15023-15027), the enzyme selects only the minor, zwitterionic ornithine with an uncharged delta-amino group for transcarbamoylation. Formation of the productive ternary complex is linked to two enzymic ionizations (pK alpha 6.2 approximately 6.3 and 9.1 approximately 9.3) and two ornithine ionizations (pK alpha 8.5 and 10.6) (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761). To elucidate the mechanism through which substrate specificity is achieved, the binding of L-ornithine to two site-specific point mutants (Arg-57----Gly and Cys-273----Ala) of the enzyme has been examined. For the Gly-57 mutant enzyme, which does not undergo the induced-fit isomerization, affinity for ornithine drops by a factor of 500. The pH profile of the apparent equilibrium constant governing the association of L-ornithine to the binary complex of this mutant reveals that only two enzymic ionizations affect ornithine binding. The ionizations linked to L-ornithine are not detected. Hence, the preisomerized binary complex binds not only poorly but also indiscriminately all ionic species of L-ornithine. For the Ala-273 mutant enzyme, which exhibits the induced-fit isomerization, affinity of the amino acid is decreased by an order of magnitude. Ionizations of L-ornithine to yield a zwitterion for binding are detected in pH analyses for this mutant, but the pK alpha of 6.2 associated with the enzymic deprotonation in the wild type is absent. Therefore, Cys-273 is a binding site of L-ornithine. The D-isomer of ornithine is a very weak, deadend ligand to all three forms of the enzyme with affinities in the millimolar range. Employing the estimated affinities of D- and L-ornithine, the binding stereospecificity of the wild-type and mutant binary complexes toward the amino acid substrate may be evaluated. L-Ornithine binds preferentially over D-ornithine by two and four orders of magnitude in the absence and presence of protein isomerization, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Chemistry, Metcalf Center for Science and Engineering, Boston University, Massachusetts 02215.
J O
JO
Goldsmith
S
S
Lee
I
I
Zambidis
L C
LC
Kuo
D002021:Buffers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000378:metabolism*; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity
D002021:Buffers; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase