Active Site Residues in Mycobacterium tuberculosis Pantothenate Synthetase Required in the Formation and Stabilization of the Adenylate Intermediate
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- 作者：Renjian Zheng ; Tarum K. Dam ; C. Fred Brewer ; and John S. Blanchard
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 8, 2004
- 年：2004
- 卷：43
- 期：22
- 页码：7171 - 7178
- 全文大小：122K
- 年卷期：v.43,no.22(June 8, 2004)
- ISSN：1520-4995
文摘
Pantothenate synthetase (EC 6.3.2.1) catalyzes the formation of pantothenate from ATP,D-pantoate, and 
-alanine in bacteria, yeast, and plants. The three-dimensional structural determination ofpantothenate 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 adenylateintermediate. To evaluate the functional roles of these strictly conserved residues, we constructed six Alamutants 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 reductionin enzyme activity, while the Q164A mutant exhibited 50-fold less activity. The rate of the isolatedadenylation reaction in single turnover studies was also reduced 40-1000-fold by the replacement of oneof these six amino acids with alanine, suggesting that these residues are essential for the formation of thepantoyl adenylate intermediate. The rate of pantothenate formation from the adenylate and -alanine inthe second half reaction could not be measured for the H44A, H47A, N69A, Q72A, and K160A mutantsand was reduced 40-fold in the Q164A mutants. The activity of the K160C mutant enzyme was markedlyenhanced by the alkylation of cysteine with bromoethylamine, further supporting the critical role of theK160 residue in pantoyl adenylate formation. Isothermal titration microcalorimetry analysis demonstratedthat the substitution of either H47 or K160 for Ala resulted in a decreased affinity of the enzyme forATP. These results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residuesare essential for the formation and stabilization of pantoyl adenylate intermediate in the pantothenatesynthetase reaction.
-alanine in bacteria, yeast, and plants. The three-dimensional structural determination ofpantothenate 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 adenylateintermediate. To evaluate the functional roles of these strictly conserved residues, we constructed six Alamutants 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 reductionin enzyme activity, while the Q164A mutant exhibited 50-fold less activity. The rate of the isolatedadenylation reaction in single turnover studies was also reduced 40-1000-fold by the replacement of oneof these six amino acids with alanine, suggesting that these residues are essential for the formation of thepantoyl adenylate intermediate. The rate of pantothenate formation from the adenylate and -alanine inthe second half reaction could not be measured for the H44A, H47A, N69A, Q72A, and K160A mutantsand was reduced 40-fold in the Q164A mutants. The activity of the K160C mutant enzyme was markedlyenhanced by the alkylation of cysteine with bromoethylamine, further supporting the critical role of theK160 residue in pantoyl adenylate formation. Isothermal titration microcalorimetry analysis demonstratedthat the substitution of either H47 or K160 for Ala resulted in a decreased affinity of the enzyme forATP. These results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residuesare essential for the formation and stabilization of pantoyl adenylate intermediate in the pantothenatesynthetase reaction.