TY - JOUR
T1 - pH and Kinetic Isotope Effects on the Reductive Half-Reaction of D-Amino Acid Oxidase
AU - Denu, John M.
AU - Fitzpatrick, Paul F.
PY - 1992/2/1
Y1 - 1992/2/1
N2 - Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pK2 values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration 15 /µM) at pH 9, D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKala values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of ~ 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H30+ and H20 below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kala value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.
AB - Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pK2 values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration 15 /µM) at pH 9, D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKala values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of ~ 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H30+ and H20 below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kala value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.
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U2 - 10.1021/bi00150a013
DO - 10.1021/bi00150a013
M3 - Article
C2 - 1356021
AN - SCOPUS:0026767696
VL - 31
SP - 8207
EP - 8215
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 35
ER -