The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis
Table 1
Kinetic and structural consequences of ASA dehydrogenase active site mutants.
Active site group
Proposed function
Mutant
Catalytic activity
Structural effect of the mutation
PDB code
Cys136 (Cys134)
Active site nucleophile
C136S
<0.01%
Rotation of S136 shifts the amino group of ASA and moves the bound phosphate by 1 Å
1PQP
His277 (His274)
Acid-base catalyst
H277N
1.0%
Breakdown of the initial tetrahedral intermediate is slowed by a shift in the phosphate position
1PQU
Arg270 (Arg267)
Binds the substrate carboxyl group
R270K R267L
0.1% 9.5%
The introduced lysine moves about 2 Å away from the original arginine position
1PS8
Glu243 (Glu240)
Binds the substrate amino group
E243D
1.2%
The bound intermediate shifts position by about 0.5 Å towards the shorter side chain of D243
1Q2X
Lys246
Helps orient bound phosphate
K246R
3.3%
The introduced arginine rotates by about 90° to form new interactions with S99 and K242
1PU2
Arg103
Binds phosphate
R103K R103L
0.4% 0.07%
Displaces the active site loop (N135 to S139) which shifts C136 away from H277
1PR3 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