Active site residues in Mycobacterium tuberculosis pantothenate synthetase required in the formation and stabilization of the adenylate intermediate.
Pantothenate synthetase (EC 6.3.2.1) catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine in bacteria, yeast, and plants. The three-dimensional structural determination of pantothenate synthetase from Mycobacterium tuberculosis has indicated specific roles for His44, His47, Asn69, Gln72, Lys160, and Gln164 residues in the binding of substrates and the pantoyl adenylate intermediate. To evaluate the functional roles of these strictly conserved residues, we constructed six Ala mutants and determined their catalytic properties. The substitution of alanine for H44, H47, N69, Q72, and K160 residues in M. tuberculosis pantothenate synthetase caused a greater than 1000-fold reduction in enzyme activity, while the Q164A mutant exhibited 50-fold less activity. The rate of the isolated adenylation reaction in single turnover studies was also reduced 40-1000-fold by the replacement of one of these six amino acids with alanine, suggesting that these residues are essential for the formation of the pantoyl adenylate intermediate. The rate of pantothenate formation from the adenylate and beta-alanine in the second half reaction could not be measured for the H44A, H47A, N69A, Q72A, and K160A mutants and was reduced 40-fold in the Q164A mutants. The activity of the K160C mutant enzyme was markedly enhanced by the alkylation of cysteine with bromoethylamine, further supporting the critical role of the K160 residue in pantoyl adenylate formation. Isothermal titration microcalorimetry analysis demonstrated that the substitution of either H47 or K160 for Ala resulted in a decreased affinity of the enzyme for ATP. These results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residues are essential for the formation and stabilization of pantoyl adenylate intermediate in the pantothenate synthetase reaction.
Zheng
Renjian
R
Departments of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Dam
Tarum K
TK
Brewer
C Fred
CF
Blanchard
John S
JS
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D017124:Conserved Sequence; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010205:Pantothenic Acid / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D010205:Pantothenic Acid; D000249:Adenosine Monophosphate; D010453:Peptide Synthases; C022318:pantothenate synthetase