Active Site Mutants of Escherichia coli Dethiobiotin Synthetase: Effects of Mutations on Enzyme Catalytic and Structural Properties

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• 作者：Guang Yang ; Tatyana Sandalova ; Karin Lohman ; Ylva Lindqvist ; and Alan R. Rendina
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：April 22, 1997
• 年：1997
• 卷：36
• 期：16
• 页码：4751 - 4760
• 全文大小：292K
• 年卷期：v.36,no.16(April 22, 1997)
• ISSN：1520-4995

文摘

Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41,implicated by the proteincrystal structure studies of Escherichia coli DTBS, weremutated to determine their function in catalysisand substrate binding. Nine mutant enzymes, T11V, E12A, E12D,K15Q, K37L, K37Q, K37R, S41A,and S41C, were overproduced in an E. coli strain lacking afunctional endogenous DTBS gene and purifiedto homogeneity. Replacement of Thr11 with valine resulted in a24 000-fold increase in theK_m(ATP)with little or no change in the K_d(ATP),K_M(DAPA) and DTBSk_cat, suggesting an essential role forthisresidue in the steady-state affinity for ATP. The two Glu12mutants showed essentially wild-type DTBSactivity (slightly elevated k_cat's).Unlike wild-type DTBS, E12A had the same apparentK_M(DAPA) atsubsaturating and saturating ATP concentrations, indicating a possiblerole for Glu12 in the binding synergybetween DAPA and ATP. The mutations in Lys15 and Lys37 resulted inloss of catalytic activity (0.01%and <0.9% of wild-type DTBS k_cat for K15Q andthe Lys37 mutant enzymes, respectively) and higherK_M's for both DAPA (40-fold and >100-foldhigher than wild-type for the K15Q and Lys37 mutantenzymes, respectively) and ATP (1800-fold and >10-fold higher thanwild-type for K15Q and the K37mutant enzymes, respectively). These results strongly suggest thatLys15 and Lys37 are crucial to bothcatalysis and substrate binding. S41A and S41C had essentially thesame k_cat as wild-type and hadmoderateincreases in the DAPA and ATP K_M andK_d (ATP) values. Replacement of Ser41 withcysteine resultedin larger effects than replacement with alanine. These datasuggest that the H-bond between N7 of DAPAand the Ser41 side chain is not very important for catalysis. Thecatalytic behavior of these mutantenzymes was also studied by pulse-chase experiments which producedresults consistent with the steady-state kinetic analyses. X-ray crystallographic studies of fourmutant enzymes, S41A, S41C, K37Q, andK37L, showed that the crystals were essentially isomorphous to that ofthe wild-type DTBS. The modelsof these mutant enzymes were well refined (1.9 -2.6 Å) and showedgood similarity to the wild-typeenzyme (rmsd of C atoms: 0.16-0.24 Å). The crystalstructure of S41C complexed with DAPA, Mn²⁺/Mg²⁺, and AMPPCP revealed a localized conformationalchange (rotations of side chains of Cys41 andThr11) which can account for the changes in the kinetic parametersobserved for S41C. The crystalstructures of the Lys37 mutant enzymes showed that the positive chargeof the side chain of Lys37 isindispensable. Mutations of Lys37 to either glutamine or leucineresulted in a shift of the metal ion (upto 0.5 Å) together with side chains of other active site residueswhich could disrupt the subtle balancebetween the positive and negative charges in the active site. Theconformational change of the phosphatebinding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide bindingobserved previously [Huang,W., Jia, J., Gibson, K. J., Taylor, W. S., Rendina, A. R., Schneider,G., & Lindqvist, Y. (1995) Biochemistry34, 10985] appears to be important to attain the proper activesite scaffold.