Mechanism of Metal-Independent Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase

Robert T. Carr, Shankar Balasubramanian, Paul C.D. Hawkins, Stephen Benkovic

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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. The ratio of alkyl to aromatic hydroxylation is very close to the ratio previously obtained [Siegmund, H., & Kaufman, S. (1991) J. Biol. Chem. 266, 2903] for this analogue and two deuterated analogues. This suggests that both RLPAH and CVPAH utilize a very similar oxygenating intermediate. We also demonstrate that both metal-free and iron-dependent enzymes hydroxylate cyclohexylalanine in a stereoselective manner.

Original languageEnglish (US)
Pages (from-to)7525-7532
Number of pages8
JournalBiochemistry
Volume34
Issue number22
DOIs
StatePublished - Jan 1 1995

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Chromobacterium
Phenylalanine Hydroxylase
Hydroxylation
Phenylalanine
Polycyclic aromatic hydrocarbons
Metals
Iron
Tyrosine
Substrates
Pterins
Biochemistry
Deuterium
Isotopes
Liver
Oxidation-Reduction
Rats
Kinetics
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Carr, R. T., Balasubramanian, S., Hawkins, P. C. D., & Benkovic, S. (1995). Mechanism of Metal-Independent Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase. Biochemistry, 34(22), 7525-7532. https://doi.org/10.1021/bi00022a028
Carr, Robert T. ; Balasubramanian, Shankar ; Hawkins, Paul C.D. ; Benkovic, Stephen. / Mechanism of Metal-Independent Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase. In: Biochemistry. 1995 ; Vol. 34, No. 22. pp. 7525-7532.
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Carr, RT, Balasubramanian, S, Hawkins, PCD & Benkovic, S 1995, 'Mechanism of Metal-Independent Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase', Biochemistry, vol. 34, no. 22, pp. 7525-7532. https://doi.org/10.1021/bi00022a028

Mechanism of Metal-Independent Hydroxylation by Chromobacterium violaceum Phenylalanine Hydroxylase. / Carr, Robert T.; Balasubramanian, Shankar; Hawkins, Paul C.D.; Benkovic, Stephen.

In: Biochemistry, Vol. 34, No. 22, 01.01.1995, p. 7525-7532.

Research output: Contribution to journalArticle

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AU - Carr, Robert T.

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N2 - 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. The ratio of alkyl to aromatic hydroxylation is very close to the ratio previously obtained [Siegmund, H., & Kaufman, S. (1991) J. Biol. Chem. 266, 2903] for this analogue and two deuterated analogues. This suggests that both RLPAH and CVPAH utilize a very similar oxygenating intermediate. We also demonstrate that both metal-free and iron-dependent enzymes hydroxylate cyclohexylalanine in a stereoselective manner.

AB - 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. The ratio of alkyl to aromatic hydroxylation is very close to the ratio previously obtained [Siegmund, H., & Kaufman, S. (1991) J. Biol. Chem. 266, 2903] for this analogue and two deuterated analogues. This suggests that both RLPAH and CVPAH utilize a very similar oxygenating intermediate. We also demonstrate that both metal-free and iron-dependent enzymes hydroxylate cyclohexylalanine in a stereoselective manner.

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