 |
PDBsum entry 1be0
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Kinetic analysis and X-Ray structure of haloalkane dehalogenase with a modified halide-Binding site.
|
|
|
Authors
|
|
G.H.Krooshof,
I.S.Ridder,
A.W.Tepper,
G.J.Vos,
H.J.Rozeboom,
K.H.Kalk,
B.W.Dijkstra,
D.B.Janssen.
|
|
|
Ref.
|
|
Biochemistry, 1998,
37,
15013-15023.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an
alkyl-enzyme intermediate. Trp175 forms a halogen/halide-binding site in the
active-site cavity together with Trp125. To get more insight in the role of
Trp175 in DhlA, we mutated residue 175 and explored the kinetics and X-ray
structure of the Trp175Tyr enzyme. The mutagenesis study indicated that an
aromatic residue at position 175 is important for the catalytic performance of
DhlA. Pre-steady-state kinetic analysis of Trp175Tyr-DhlA showed that the
observed 6-fold increase of the Km for 1,2-dibromoethane (DBE) results from
reduced rates of both DBE binding and cleavage of the carbon-bromine bond.
Furthermore, the enzyme isomerization preceding bromide release became 4-fold
faster in the mutant enzyme. As a result, the rate of hydrolysis of the
alkyl-enzyme intermediate became the main determinant of the kcat for DBE, which
was 2-fold higher than the wild-type kcat. The X-ray structure of the mutant
enzyme at pH 6 showed that the backbone structure of the enzyme remains intact
and that the tyrosine side chain lies in the same plane as Trp175 in the
wild-type enzyme. The Clalpha-stabilizing aromatic rings of Tyr175 and Trp125
are 0.7 A further apart and due to the smaller size of the mutated residue, the
volume of the cavity has increased by one-fifth. X-ray structures of mutant and
wild-type enzyme at pH 5 demonstrated that the Tyr175 side chain rotated away
upon binding of an acetic acid molecule, leaving one of its oxygen atoms
hydrogen bonded to the indole nitrogen of Trp125 only. These structural changes
indicate a weakened interaction between residue 175 and the halogen atom or
halide ion in the active site and help to explain the kinetic changes induced by
the Trp175Tyr mutation.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase.
|
|
|
Authors
|
|
K.H.Verschueren,
F.Seljée,
H.J.Rozeboom,
K.H.Kalk,
B.W.Dijkstra.
|
|
|
Ref.
|
|
Nature, 1993,
363,
693-698.
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
Secondary reference #2
|
|
|
Title
|
|
Refined X-Ray structures of haloalkane dehalogenase at ph 6.2 and ph 8.2 and implications for the reaction mechanism.
|
|
|
Authors
|
|
K.H.Verschueren,
S.M.Franken,
H.J.Rozeboom,
K.H.Kalk,
B.W.Dijkstra.
|
|
|
Ref.
|
|
J Mol Biol, 1993,
232,
856-872.
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
Secondary reference #3
|
|
|
Title
|
|
Crystal structure of haloalkane dehalogenase: an enzyme to detoxify halogenated alkanes.
|
|
|
Authors
|
|
S.M.Franken,
H.J.Rozeboom,
K.H.Kalk,
B.W.Dijkstra.
|
|
|
Ref.
|
|
Embo J, 1991,
10,
1297-1302.
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
Secondary reference #4
|
|
|
Title
|
|
Crystallization of haloalkane dehalogenase from xanthobacter autotrophicus gj10.
|
|
|
Authors
|
|
H.J.Rozeboom,
J.Kingma,
D.B.Janssen,
B.W.Dijkstra.
|
|
|
Ref.
|
|
J Mol Biol, 1988,
200,
611-612.
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
