Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase.
Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing a tetrahydrobiopterin cofactor. Several key mechanistic questions have yet to be resolved, specifically the identity of the hydroxylating species and the role of the non-heme iron which is present in all of the mammalian PAHs. Recently, we have demonstrated that a bacterial PAH from Chromobacterium violaceum does not require any redox active metal for activity [Carr, R. T., & Benkovic, S. J. (1993) Biochemistry 32, 14132-14138]. To identify the function of iron in the mammalian PAH's, we have undertaken a series of experiments to compare the mechanisms of this metal-independent PAH with the iron-dependent PAH from rat liver. Using [4-2H]phenylalanine as a substrate gave a kinetic isotope effect on hydroxylation of unity for CVPAH which is in agreement with previous values reported for RLPAH. The [4-2H]phenylalanine underwent an NIH shift upon hydroxylation by CVPAH. The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both RLPAH and CVPAH using [4-2H]phenylalanine and [2,3,5,6-2H]phenylalanine as substrates. This suggests that PAH from either source probably does not directly mediate the NIH shift mechanism. No uncoupled pterin turnover was observed for CVPAH with either L-tyrosine or p-chloro-L-phenylalanine as substrate or tetrahydropterin as cofactor, each of which causes uncoupled turnover with RLPAH. CVPAH readily accepts 4-methylphenylalanine as a substrate giving 4-(hydroxymethyl)phenylalanine as the major product and 3-methyltyrosine as the only other minor product.(ABSTRACT TRUNCATED AT 250 WORDS)
Carr
R T
RT
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Balasubramanian
S
S
Hawkins
P C
PC
Benkovic
S J
SJ
D000818:Animals; D002861:Chromobacterium / Q000201:enzymology*; D003001:Cloning, Molecular; D003903:Deuterium; D004926:Escherichia coli; D006900:Hydroxylation; D007501:Iron / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008667:Metalloproteins / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D019159:Nonheme Iron Proteins; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D008667:Metalloproteins; D008670:Metals; D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D003903:Deuterium; D007501:Iron; D010651:Phenylalanine Hydroxylase