Protocatechuate 3,4-dioxygenase (EC 184.108.40.206) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into β-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, Mössbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.
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We would like to acknowledge the contributions of Dr. W.H. Orme-Johnson of the University of Wisconsin for providing valuable advice and making his EPR spectrometer available. We thank Dr. P.J. Chapman for generously providing the fluorinated compounds, Dr. R.G. Bryant and Dr. R. Zimmermann for valuable discussions, Mr. W. Hamilton for capable technical assistance with the EPR spectrometer, and Mr. S. Pratt for experimental assistance in organic synthesis. This work was supported by USPHS Grant GM 22701, NSF Grant PCM-17318, and by a Research Career Development Award K04-GM70683 (E.M.).