Mössbauer, Electron‐Paramagnetic‐Resonance and X‐ray‐Absorption Fine‐Structure Studies of the Iron Environment in Recombinant Human Tyrosine Hydroxylase

Meyer‐Klaucke, Wolfram; Winkler, H.; Schünemann, Volker; Trautwein, Alfred X.; Nolting, H.‐F.; Haavik, Jan

doi:10.1111/j.1432-1033.1996.00432.x

Cited by 45 publications

(58 citation statements)

References 32 publications

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“…Recent physicochemical studies focusing on the redox states of iron incorporated into TH molecule have clarified precisely the mechanisms by which dopamine chelates iron at the active site of the enzyme. In addition, the trapping of the active site iron in the ferric state, the binding mode of 6RBPH4 with iron, and the reduction of ferric iron by 6RBPH4 during catalytic turnover have been extensively characterized (Michaud-Soret et al, 1995;Martinez et al, 1996;Meyer-Klaucke et al, 1996;Ramsey et al, 1996). More recently, the differential binding of dopamine to the ferric and ferrous state TH was clearly demonstrated (Ramsey and Fitzpatrick, 1998).…”

Section: Discussionmentioning

confidence: 99%

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme

Nakashima¹,

Mori²,

Suzuki³

et al. 1999

Journal of Neurochemistry

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Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 3 5 , 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type l were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus-deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(~-erythro-l',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (GRBPH,) among the four enzymes. The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del-38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor GRBPH, dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence G l~~~-A r g~~-A r g~~ plays a key role in determining the competition between dopamine and GRBPH, and affects the efficiency of dopamine inhibition of the catalytic activity. Key Words: Tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydr0pterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.21, which catalyzes the conversion of L-DOPA from L-tyrosine (Nagatsu et al., 1964), is a rate-limiting enzyme in the biosynthesis of catecholamines (Levitt et al., 1965). The enzyme requires a reduced pterin (Nagatsu et al., 1964), and (6R)-(~-erythro-1 ,2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tebxhydropteridine (6R-tebxhydrobiopteri~ 6RBPH4) is the natural cofactor (Kaufman, 1963;Brenneman and Kaufman, 1964;Matsuura et al., 1985). TH also requires ferrous iron (Nagatsu et al., 1964;Shiman et al., 1971). TH consists of a catalytic domain (C-domain) and a regulatory domain (R-domain) (Abate and Joh, 1991). The C-domain is located at the C-terminal two-thirds of the molecule and binds the substrates (L-tyrosine and molecular oxygen) and the cofactor (6RBPH,). In contrast, the R-domain has been assigned to the N-terminal end (Hoeldtke and Kaufman, 1977;Abate et al., 1988;Abate and Joh, 1991) and has an important role in substrate specificity and in control of the catalytic activ...

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Section: Discussionmentioning

confidence: 99%

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme

Nakashima¹,

Mori²,

Suzuki³

et al. 1999

Journal of Neurochemistry

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“…Therefore the specific activity shown in Fig. 3 was fitted to Equation 13 for k cat(app) given under "Experimental Procedures." The results are given in Table III.…”

Section: Mono-and Binuclear Zn 2ϩ -␤-Lactamasementioning

confidence: 99%

Mono- and Binuclear Zn2+-β-Lactamase

Paul-Soto¹,

Bauer²,

Frère³

et al. 1999

Journal of Biological Chemistry

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127

128

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When expressed by pathogenic bacteria, Zn2؉ -␤-lactamases induce resistance to most ␤-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn 2؉ -␤-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn 2؉ -␤-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn 2؉ (mononuclear) and di-Zn 2؉ (binuclear) Zn 2؉ -␤-lactamases are catalytically active but with different kinetic properties. The mono-Zn2؉ -␤-lactamase requires the conserved cysteine residue for hydrolysis of the ␤-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.

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“…The K-edge XAFS data were collected at beam line D2 at the European Molecular Biology Laboratory Outstation Hamburg with a positron beam energy of 4.6 GeV and a maximum stored current of 100 mA in fluorescence mode [Meyer-Klaucke et al (1996)]. The samples were measured as frozen solutions at 18 K. An energy resolution of better than 2.5 eV has been achieved.…”

Section: Methodsmentioning

confidence: 99%

A Comparison of Bacillus Cereus and Aeromonas Hydrophilia Zn-β-lactamases

Meyer‐Klaucke¹,

Soto²,

Hernandez-Valladares³

et al. 1999

J Synchrotron Radiat

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We have performed EXAFS studies on the B. cereus 5/B/6 and A. hydrophila enzymes, both at pH 6.5 containing about 1 equivalent of Zn. Note that the B. cereus enzymes from strains 5/B/6 and 568/H/9 differ in 17 aminoacid substitutions; none of which is located at the active site. Therefore, although the crystal structure of the 5/B/6 enzyme is not known similar metal coordination patterns are expected. For the A. hydrophila enzyme no crystal structure is available at present. Laboratory, Outstation Hamburg, Notkestr. 85, 22603 Hamburg, Germany, bFachdchtung 12.4 In an increasing number of pathogenic bacteria Zn-13-1actamases are being discovered as major factors of antibiotic resistance. For a possible strategy to fight these bacteria, it is important to understand the catalytic mechanism. X-ray absorption spectroscopy measurements on the zinc-K-edge of 13-1actamases from Bacillus cereus (strain 5/B/6) and Aeromonas hydrophila (AE 036) gives evidence for differences in the mode of zinc sulfur coordination and information about the coordination to N and O donor ligands. "European Molecular Biology

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Mössbauer, Electron‐Paramagnetic‐Resonance and X‐ray‐Absorption Fine‐Structure Studies of the Iron Environment in Recombinant Human Tyrosine Hydroxylase

Cited by 45 publications

References 32 publications

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme

Mono- and Binuclear Zn2+-β-Lactamase

A Comparison of Bacillus Cereus and Aeromonas Hydrophilia Zn-β-lactamases

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