Re-evaluation of EDTA-Chelated Biuret Reagent

Chromý, V.; Fischer, J.; Kulhánek, V

doi:10.1093/clinchem/20.10.1362

Cited by 51 publications

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“…vinelandii cells and purified to homogeneity as described (Seefeldt & Mortenson, 1993). Protein concentrations were determined by the modified biuret method using bovine serum albumin as standard (Chromy et al, 1974). All proteins used were homogeneous as judged by analysis on SDS gels stained with Coomassie blue (Seefeldt et al, 1992).…”

Section: Site-directed Mutagenesis Expression and Purification Of Amentioning

confidence: 99%

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Seefeldt

1994

Protein Science

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Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A . vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments. In addition, it was found that changing Arg 140 and Lys 143 to Gln resulted in a significant uncoupling of MgATP hydrolysis from intercomponent electron transfer rates between FeP and MoFeP. The wild-type complex was found to hydrolyze 2.5 MgATP per electron transferred, whereas the R140Q and K143Q proteins, when coupled to wild-type MoFeP, required 6 and 31 MgATP for each electron transferred, respectively. It is concluded that both Arg 140 and Lys 143 of the A . vinelandii nitrogenase FeP are important in the docking interaction with the MoFeP and that these residues probably function in aligning the FeP with the MoFeP for intercomponent electron transfer. A model is discussed in which these positively charged amino acids of the FeP might interact with negatively charged amino acids (Asp and Glu) on the MoFeP near its 8Fe cluster.Keywords: electron transfer; iron protein; molybdenum-iron protein; MgATP hydrolysis; nitrogenase; protein docking; site-directed mutagenesis Biological dinitrogen reduction is catalyzed by nitrogenase, a complex metalloenzyme consisting of the 2 separable component proteins, the molybdenum-iron protein and the iron protein (current reviews: Burris, 1991;Smith & Eady, 1992;Dean et al., Reprint requests to: Lance C . Seefeldt, Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300; e-mail: seefeldt@cc.usu.edu.Abbreviations: FeP, nitrogenase iron protein; MoFeP, nitrogenase molybdenum-iron protein; FeP-MoFeP, iron protein-molybdenumiron protein comple...

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Section: Site-directed Mutagenesis Expression and Purification Of Amentioning

confidence: 99%

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Seefeldt

1994

Protein Science

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“…SDS gel analysis of proteins and N-terminal amino acid sequencing of the altered FePs was accomplished as described previously (Seefeldt et al, 1992). Protein was determined by the modified Biuret method (Chromy et al, 1974) using bovine serum albumin as standard.…”

Section: Protein Determination Sds Gel Electrophoresis and N-terminmentioning

confidence: 99%

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn²⁺ to show interaction of serine 16 with Mg²⁺

Seefeldt

Mortenson

1993

Protein Science

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MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the y-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A . vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured K,,, of 20 pM compared to the wild-type FeP K,,, of 220 pM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kca,/K,,,) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP. The S16T FeP was severely cornpromised in its interaction with MnATP, suggesting a mechanism where the hydroxyl group of amino acid 16 interacts with the Mgz+ of bound MgATP.

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“…Analytical Procedures. Protein concentrations were determined by means of a modified biuret assay using bovine serum albumin as a standard (32). Metal analysis were performed by ICP-MS at the Utah State University Veterinary Diagnostics Laboratory and by the PAR/PMPS assay modified by Krum et al (7,9).…”

Section: Methodsmentioning

confidence: 99%

Evidence for a Metal−Thiolate Intermediate in Alkyl Group Transfer from Epoxypropane to Coenzyme M and Cooperative Metal Ion Binding in Epoxyalkane:CoM Transferase

Boyd

Ensign

2005

Biochemistry

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Epoxyalkane:coenzyme M transferase (EaCoMT) catalyzes the nucleophilic addition of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) to epoxypropane forming 2-hydroxypropyl-CoM. The biochemical properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. The enzyme has a hexameric (alpha(6)) structure with one zinc atom per subunit. In the present work M(2+) binding and the role of Zn(2+) in EaCoMT have been established through a combination of biochemical, calorimetric, and spectroscopic techniques. A variety of metal ions, including Zn(2+), Co(2+), Cd(2+), and Ni(2+), were capable of activating a Zn-deficient "apo" form of EaCoMT, affording enzymes with various levels of activity. Titration of Co(2+) into apo-EaCoMT resulted in UV-visible spectroscopic changes consistent with the formation of a tetrahedral Co(2+) binding site, with coordination of bound Co(2+) to two thiolate ligands. Quantification of UV-visible spectral changes upon Co(2+) titration into apo-EaCoMT demonstrated that EaCoMT binds Co(2+) cooperatively at six interacting sites. Isothermal titration calorimetric studies of Co(2+) and Zn(2+) binding to EaCoMT also showed cooperativity for metal ion binding among six sites. The addition of CoM to Co(2+)-substituted EaCoMT resulted in UV-visible spectral changes indicative of formation of a new thiol-Co(2+) bond. Co(2+)-substituted EaCoMT exhibited a unique Co(2+) EPR spectrum, and this spectrum was perturbed significantly upon addition of CoM. The presence of a divalent metal ion was required for the release of protons from CoM upon binding to EaCoMT, with Zn(2+), Co(2+), and Cd(2+) each facilitating proton release. The divalent metal ion of EaCoMT is proposed to play a key role in the coordination and deprotonation of CoM, possibly through formation of a metal-thiolate that is activated for attack on the epoxide substrate.

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Re-evaluation of EDTA-Chelated Biuret Reagent

Cited by 51 publications

References 0 publications

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn²⁺ to show interaction of serine 16 with Mg²⁺

Evidence for a Metal−Thiolate Intermediate in Alkyl Group Transfer from Epoxypropane to Coenzyme M and Cooperative Metal Ion Binding in Epoxyalkane:CoM Transferase

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Re-evaluation of EDTA-Chelated Biuret Reagent

Cited by 51 publications

References 0 publications

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn2+ to show interaction of serine 16 with Mg2+

Evidence for a Metal−Thiolate Intermediate in Alkyl Group Transfer from Epoxypropane to Coenzyme M and Cooperative Metal Ion Binding in Epoxyalkane:CoM Transferase

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Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn²⁺ to show interaction of serine 16 with Mg²⁺