SELECTIVE MODIFICATION OF ALKYLAMMONIUM ION SPECIFICITY IN TRIMETHYLAMINE DEHYDROGENASE BY THE RATIONAL ENGINEERING OF CATION-PI BONDING

In trimethylamine dehydrogenase (TMADH), substrate is bound in the act ive site by organic cation-pi bonding mediated by residues Tyr-60, Trp -264, and Trp-355. In the closely related dimethylamine dehydrogenase (DMADH), modeling suggests that a mixture of cation-pi bonding and con ventional hydrogen bonding is responsible for binding dimethylamine. T he active sites of both enzymes are highly conserved, but three change s in amino acid identity (residues Tyr-60 --> Gln, Ser-74 --> Thr, and Trp-105 --> Phe, TMADH numbering) were identified as probable determi nants for tertiary --> secondary alkylammonium ion specificity. In an attempt to switch the substrate specificity of TMADH so that the enzym e operates more efficiently with dimethylamine, three mutant proteins of TMADH were isolated. The mutant forms contained either a single mut ation (Y60Q), double mutation (Y60Q . S74T) or triple mutation (Y60Q . S74T . W105F). A kinetic analysis in the steady state with trimethyla mine and dimethylamine as substrate indicated that the specificity of the triple mutant was switched approximately 90 000-fold in favor of d imethylamine. The major component of this switch in specificity is a s elective impairment of the catalytic efficiency of the enzyme with tri methylamine. Rapid-scanning and single wavelength stopped-flow spectro scopic studies revealed that the major effects of the mutations are on the rate of flavin reduction and the dissociation constant for substr ate when trimethylamine is used as substrate. With dimethylamine as su bstrate, the rate constants for flavin reduction and the dissociation constants for substrate are not substantially affected in the mutant e nzymes compared with wild-type TMADH. The results indicate a selective modification of the substrate-binding site in TMADH (that impairs cat alysis with trimethylamine but not with dimethylamine) is responsible for the switch in substrate specificity displayed by the mutant enzyme s.