Tertiary structure and mutagenesis studies

TKase is a homodimeric protein with a subunit of about 70 kDa. The X-ray structures of TKase of E. coli,62 S. cerevisiae,61-63ā€”67 Leishmania mexicana6& and mize69 have been solved. In addition, the crystal structures of a number of site-directed mutants have been determined.70-73 Schneider and co-workers have reported a series of studies in which they have mutated important residues of active site of TKase to elucidate the reaction mechanism and explain the origin of the stere-ospecificity of the Cā€”C bond-forming process (Table 7).74ā€”76 The conserved residue Asp477 of yeast TKase is located in the substrate channel of the enzyme and forms a hydrogen bond with the C2-hydroxyl group of the acceptor substrate as shown in Fig. 19. In the wild-type enzyme, the kcat/Km values are 103-104 lower for 2-deoxyaldoses and L-configured substrate. In the Asp477Ala mutant, the kcat/Km values for D-a-hydroxyaldehydes are lower by 103, while the kcat/Km values for the l- or 2-deoxy aldehydes are similar to that of the wild-type enzyme.77 These results indicate that Asp477 is involved in determining the enantioselectivity of TKase.

Table 7

Available crystal Structures of transketolase

Enzyme source and species

References

Table 7

Available crystal Structures of transketolase

Enzyme source and species

Escherichia coli

Enzyme-ThDP

1.9

40

Mize

Enzyme-ThDP

2.3

69

Leishmania mexicana

Enzyme-ThDP

2.2

68

Yeast

Holoenzyme

2.0

63,64

Apoenzyme

2.8

65

Enzyme-6' -methyl-ThDP

2.3

66

Enzyme-N1-ThDP

2.7

61

Enzyme-N3-ThDP

2.7

61

Holoenzyme-erythrose-4-phosphate

2.3

61

E418A

2.4

67

E418Q

2.9

70

H103A

2.3

70

H103N

2.7

71

E162Q

2.7

71

D382A

2.9

72

H263A

2.8

72

2.6

73

Figure 19: Active site of transketolase.

Substrate-binding site

His481 h N,

Enantiospecificity ^ ^^

Arg528

Phosphate-binding site

NH H NH

Ser386

HN-His30 His263

Glu418

His69

His103

Substrate-binding site

Figure 19: Continued.

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