Table 1: Kinetic and structural consequences of ASA dehydrogenase active site mutants.

Active site group Proposed functionMutantCatalytic activity Structural effect of the mutationPDB code

Cys136 (Cys134)Active site nucleophileC136S<0.01%Rotation of S136 shifts the amino group of ASA and moves the bound phosphate by 1 Å1PQP

His277 (His274)Acid-base catalystH277N1.0%Breakdown of the initial tetrahedral intermediate is slowed by a shift in the phosphate position1PQU

Arg270 (Arg267)Binds the substrate carboxyl groupR270K
R267L
0.1%
9.5%
The introduced lysine moves about 2 Å away from the original arginine position1PS8

Glu243 (Glu240)Binds the substrate amino groupE243D1.2%The bound intermediate shifts position by about 0.5 Å towards the shorter side chain of D2431Q2X

Lys246Helps orient bound phosphateK246R3.3%The introduced arginine rotates by about 90° to form new interactions with S99 and K2421PU2

Arg103Binds phosphateR103K
R103L
0.4%
0.07%
Displaces the active site loop (N135 to S139) which shifts C136 away from H2771PR3
1OZA

H. influenzae sequence numbering, with the numbers in parenthesis referring to the V. cholerae sequence
determined by varying ASA at fixed NADP levels and expressed as a percent of wild type enzyme activity