Haloalkane dehalogenases: steady-state kinetics and halide inhibition.
The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.
Schindler
J F
JF
Chemical Sciences and Technology Division, Bioscience and Biotechnology Group, Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Naranjo
P A
PA
Honaberger
D A
DA
Chang
C H
CH
Brainard
J R
JR
Vanderberg
L A
LA
Unkefer
C J
CJ
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002712:Chlorides / Q000737:chemistry* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005023:Ethylene Chlorohydrin / Q000737:chemistry / Q000494:pharmacology; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives; D010649:Phenylalanine / Q000235:genetics; D012240:Rhodococcus / Q000201:enzymology; D013379:Substrate Specificity; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D002712:Chlorides; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009596:Nitrophenols; D014443:Tyrosine; D010649:Phenylalanine; D005023:Ethylene Chlorohydrin; C039339:3-cresol purple; D014364:Tryptophan; D006867:Hydrolases; C046137:haloalkane dehalogenase; D010637:Phenolsulfonphthalein; C049017:3-nitrophenol