Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct.
Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.
Mure
Minae
M
Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045, USA. mmure@ku.edu
Kurtis
Christian R
CR
Brown
Doreen E
DE
Rogers
Melanie S
MS
Tambyrajah
Winston S
WS
Saysell
Colin
C
Wilmot
Carrie M
CM
Phillips
Simon E V
SE
Knowles
Peter F
PF
Dooley
David M
DM
McPherson
Michael J
MJ
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001391:Azo Compounds / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004795:Enzyme Stability / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011728:Pyridones / Q000737:chemistry*; D012118:Resorcinols / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000235:genetics
D001391:Azo Compounds; D002413:Cations, Divalent; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011728:Pyridones; D012118:Resorcinols; D001224:Aspartic Acid; D003035:Cobalt; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; C062607:2-hydrazinopyridine; D001216:Asparagine; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); C018021:cobaltous chloride; C028398:4-(2-pyridylazo)resorcinol; C029892:cupric chloride