Abstract
Expression conditions in Escherichia coli of wild-type, Y224F, and Y228F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties similar to those of the wild-type enzyme, in all oxidation-reduction states. All enzymes were studied by steady state and rapid reaction methods. Turnover numbers determined for Y224F DAAO with different substrates were similar to those of wild-type protein, while the Y228F DAAO always showed lower turnover numbers and higher Km values for the D-amino acid. Analyses of reduction traces at 450 and 550 nm of stopped-flow experiments with wild-type DAAO showed the presence of a new phase, the conversion between two different charge-transfer complexes of the reduced enzyme and imino acid product. The substitution of Tyr-228 totally abolished the formation of the long wavelength bands while Y224F DAAO showed long wavelength absorbance only for the first intermediate. Reoxidation of the reduced flavin results from reaction of oxygen with the first charge-transfer complex. The rate of reduction with D-alanine as substrate was 1225,45 and 10 s-1 for wild-type, Y224F, and Y228F DAAOs, respectively. Comparison of the properties of these two mutant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-reaction of the enzyme.
Highlights
Y224F, and Y228F mutants of pig kidney D-amino acid abstraction from the D-amino acid (Walsh et al, 1973; Porter et oxidase (DAAO) have been changed to yield more en- al., 1977) or through a-proton abstractionconcerted with eleczyme
Comparison of the properties of these two muetann-t quences. This analysis shows that Tyr-228 and His-307, both zyme forms with those of the wild-type DAAO indicate identified by the partial inactivationof the enzyme by proparthat both tyrosine residues have their main function ignylglycine (Rudieet al.,1980; Ronchi et al, 1982), are theonly the reductive half-reaction of the enzyme
After a further h, the cells were collected by centrifugation, and a crude extract was prepared as described under "ExperimentalProcedures." DAAO activity was determined in the crude extract employing the o-phenylglycine enazsysmaye and the total protein concentration usingthe BCA protein assay. 1 unit is defined as the amountof enzyme that converts 1 pmol of D-phenylglycinein 1 min at "C(Fonda and Anderson1, 967)
Summary
Specific activity mixture containing 100 mM sodium pyrophosphate, pH 8.5, 12.5 mM. 0.05 rity of the enzyme was determined by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). At. The dissociation constant for sulfite was measured by equilibrium different times after induction, cells were collected and the titrations by measuring theloss of visible absorbance due to formation specific activities of the extracts after sonication were deterof the flavin N6-sulfite adduct (Massey et al, 1969). Higher activity and higherspecificactivity was constant for the complex of holoenzyme with anthranilate was esti- obtained by induction at higher cell density (OD,, = 2.8) and at mated according to Benesi and Hildebrand (1949fr)om the changes in the absorbance spectrumof the enzyme on addition of the inhibitor. University of Michi- reported in Table 11.The best result oisbtained at high inducer gan), which permits the analyofsitshe databy exponential fits basedon concentrations, and enzyme production appears to be almost the Marquardt algorithm Y224F mutant, while for the Y228F mutant, because of its low catalyticactivitywith the D-phenylglycine assay used, the
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
