Cobamides and ribonucleotide reduction. VII. Cob(II)alamin as a sensitive probe for the active center of ribonucleotide reductase

Hamilton, John A.; Yamada, Ryohei; Blakley, Raymond L.; Hogenkamp, Harry P. C.; Looney, F.D.; Winfield, M.E.

doi:10.1021/bi00778a023

Cited by 58 publications

(48 citation statements)

References 20 publications

(31 reference statements)

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“…Thus, the presence of a stoichiometric amount of adenosine, at least at the concentrations of adenosine used (1-20 M), lowered the K i of the cob(II)alamin inhibition. The result that cob(II)alamin binds tightly is fully consistent with the knowledge that the corrin half of the posthomolysis intermediate is cob(II)alamin, and the idea that tight binding of cob(II)alamin and 5Ј-deoxyadenosine provides an important part of the driving force for homolytic cleavage of the Co-C bond (12,30,41).…”

Section: Methodssupporting

confidence: 81%

“…To generate cob(II)alamin, aquocobalamin was reduced by an excess of thiol to cob(II)alamin (28)(29)(30) under the assay conditions (30 mM DTT, pH 7.3); aquocobalamin was completely converted to cob(II)alamin by dithiothreitol within 1 min, and the resultant cob(II)alamin was stable for the duration of the assay period. Cob(II)alamin inhibition was evaluated in the absence and presence of an equivalent amount of adenosine in the assay solution, entries 6 and 7 in Table 1, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

Suto¹,

Poppe

Rétey

et al. 1999

Bioorganic Chemistry

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Section: Methodssupporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

Suto¹,

Poppe

Rétey

et al. 1999

Bioorganic Chemistry

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“…48,49 As outlined subsequently, the exchange reaction has played a very important role in defining the catalytic capabilities of RTPR.…”

Section: Class II Ribonucleotide Reductasesmentioning

confidence: 99%

Protein Radicals in Enzyme Catalysis

1998

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ContentsI. Introduction 705 II. General Principles 706 A. One Electron Oxidized Amino Acids Thus Far Identified in Proteins 706 B. Biosynthesis of (Modified) Amino Acid Radicals 706 C. Emerging General Chemical Properties of Enzymes Utilizing Protein Radicals for Catalysis 708 D. Methods To Examine Radical Dependent Reactions 710 III. Background on Ribonucleotide Reductases 710 IV. Class I Ribonucleotide Reductases 712 A. Formation of the Tyrosyl Radical 713 B. Function of the Tyrosyl Radical 714 C. Thiyl Radical Involvement in Catalysis 716 D. Evidence for Enzyme-Mediated Radical Chemistry Using Nucleotide Analogues 717 E. Structure 719 V. Class II Ribonucleotide Reductases 719 A. Exchange Reaction: Detection of the Elusive Thiyl Radical 719 B. Role of the Thiyl Radical in Catalysis 721 VI. Pyruvate Formate Lyase 722 A. Characterization of the Glycyl Radical 723 B. Formation of the Glycyl Radical 723 C. Catalytic Mechanism 724 D. Enzyme-Mediated Radical Chemistry with Pyruvate Analogues 727 VII. Anaerobic Ribonucleotide Reductase 728 A. Background 728 B. Requirement for a Glycyl Radical 728 C. Mechanism of Nucleotide Reduction 729 VIII. Cytochrome c Peroxidase 730 A. Formation of the Ferryl Heme/Tryptophan Cation Radical 730 B. Proposed Function of the Tryptophan Radical in Electron Transfer 731 IX. Prostaglandin H Synthase 733 A. Structure: A Tyrosine Residue Revealed 733 B. Proposed Role of the Tyrosyl Radical in Catalysis 734 X. Photosynthetic Oxygen Evolution 736 A. Background 736 B. Proposed Roles of Tyrosyl Radicals Y Z • and Y D • 737 C. Spectroscopic Studies on Y Z • and Y D • 738 D. Proposed Mechanisms for Water Oxidation and O 2 Evolution 739 XI. Galactose Oxidase 741 A. Different Redox States of Galactose Oxidase 741 B. Structure: A Modified Tyrosine Residue Revealed 742 C. Is the Oxidized Amino Acid Associated with Apo Oxidized GAO the Novel Ortho-Thiol-Substituted Tyrosine? 743 D. Catalytic Mechanism 743 XII. Quinoproteins 744 A. Copper-Dependent Amine Oxidases 744 B. Methylamine Dehydrogenase 749 XIII. Other Systems in Which Protein-Based Radicals Have Been Proposed or Detected 751 A. Bovine Liver Catalase 751 B. DNA Photolyase 751 C. Dopamine β Monooxygenase 752 XIV. Summary and Outlook 754 XV. Abbreviations 754 XVI. Acknowledgments 755 XVII. References 755 JoAnne Stubbe is the Novartis professor of Chemistry and Biology at the Massachusetts Institute of Technology. She received her undergraduate degree from the University of Pennsylvania working with Ed Thornton and did NSF-sponsored undergraduate research with Ed Trachtenberg at Clark University. These mentors played a strong role in her interest in physical organic chemistry. She received her Ph.D.

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“…In the latter enzyme cob(1I)-alamin also represents an inactive enzyme-coenzyme complex. Inactivation of adenosylcobalamin-dependent ribnnucleotide reductase is also accompanied by the conversion of adenosylcobalamin to cob(I1)alamin in the presence of the substrate (Hamilton et al, 1971;Yamada et al, 1971).…”

Section: Inactivation Of Glutamate Mutase Under the Formation Of Cob(mentioning

confidence: 99%

Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

Zelder¹,

Beatrix²,

Leutbecher³

1994

European Journal of Biochemistry

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The glutamate mutase dependent on adenosylcobalamin (coenzyme B,J catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S, 3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer E~ ( E , 53.5 kDa) and S, a monomer (a, 14.8 ma). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins.Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme BIZ. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B,, resulted in the formation of a fully active glutamate mutase heterotetramer (~~a~) containing one molecule of coenzyme BIZ.EPR spectra of recombinant glutamate mutase, now available in sufficiently large amounts, were recorded after incubation of the enzyme with coenzyme B,, and (S)-glutamate. The EPR signals (gx, = 2.1, g, = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(I1)alamin (gx,, = 2.25). The spin concentration was 34-50% of the concentration of the enzyme ( E~C T J coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)- [2,3,3,4,]glutaate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7 %. These data suggest an involvement of the Co(I1)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the perdeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species. It is discussed that the paramagnetic species represents a radical pair composed of Co(I1) and an organic radical.Overnight incubation of glutamate mutase and coenzyme with substrate or one of the competitive inhibitors resulted in an inactive enzyme and cob(II)alamin, which was identified by EPR spectroscopy (gX,, = 2.25). This cob(I1)alamin appeared to be very similar to a cob(I1)amide present in inactive glutamate mutase preparations...

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Cobamides and ribonucleotide reduction. VII. Cob(II)alamin as a sensitive probe for the active center of ribonucleotide reductase

Cited by 58 publications

References 20 publications

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

Protein Radicals in Enzyme Catalysis

Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

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Cobamides and ribonucleotide reduction. VII. Cob(II)alamin as a sensitive probe for the active center of ribonucleotide reductase

Cited by 58 publications

References 20 publications

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

Protein Radicals in Enzyme Catalysis

Characterization of the Coenzyme‐B12–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

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Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli