Magnetic resonance studies of enzymesubstrate complexes with paramagnetic probes as illustrated by creatine kinase

Cohn, Mildred

doi:10.1017/s0033583500004418

Cited by 63 publications

(15 citation statements)

References 46 publications

(63 reference statements)

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“…The conformation of tRNAf1Met in solution is monitored by the paramagnetic contribution of the spin label to the line broadening of proton resonances. The broadening is a function of the distance between the covalently bound nitroxide spin label and specific protons (15). Observation of resolved resonances in NMR spectra of tRNA is limited to a "window" in the methyl region and to the low field region of slowly exchanging N-bonded hydrogens below 11 ppm.…”

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confidence: 99%

Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET.

Daniel¹,

Cohn²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Thiouridine at position 8 (s4U8) of tRNAf-Met was spin-labeled with the nitroxide free radical, N-1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) bromacetamide, for proton nuclear magnetic resonance spectroscopic studies. The wellresolved methyl peak of ribothymidine is unperturbed, but the peak tentatively assigned to the C-5 methylene group of dihydrouridine is considerably broadened in spin-labeled tRNAf Met, Of the approximately 27 slowly exchanging protons observed in the region between 11 and 15 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid, the equivalent of about five protons apparently disappeared in spin-labeled tRNAf1Met. The well-resolved single proton at 14.8 ppm was missing not only in the paramagnetic species, but also in the diamagnetic reduced form of spin-labeled tRNAfMet, and was unequivocally identified as a hydrogen bond involving s4U8 by comparison of several forms of tRNAf Met specifically modified at s4U. Evidence that the perturbation of a second single proton resonance at 14.6 ppm (shift and broadening) is coupled to the loss of a tertiary hydrogen bond involving residue 8, arises from the same modified forms. The resolved resonances in the methyl and N-H regions, particularly the resonance at 14.6 ppm as well as the four N-bonded proton resonances at higher field which are broadened solely due to their proximity to the unpaired electron of the spin label, provide specific indicators of the geometry of tRNAfIMt structure in solution. Their observability by nuclear magnetic resonance spectroscopy opens up the possibility of monitoring distance changes among the base residues of spin-labeled tRNAf Met upon its interaction with aminoacyl-tRNA synthetase an other enzymes.The mechanism of recognition of specific tRNAs by their cognate aminoacyl-tRNA synthetases has been the subject of many investigations. For example, the relation of specificity to primary structure has been investigated by (i) comparison of sequences for homology in a group of tRNAs that are active substrates for a given synthetase (1, 2) and (ii) modifications of primary structure to distinguish essential from nonessential residues (3). Although the results have been suggestive, no universal principles of recognition have emerged. The conclusion that a specific tertiary structure of the molecule is necessary, if not sufficient, for recognition was early suggested by Fresco et al. (4) and emphasized by Cramer (1) and is now inescapable from the overwhelming amount of accumulated data.The three-dimensional structure derived from x-ray crystallographic analysis of yeast tRNAPhe (5,6) may prove to have specific as well as universal (7) The electron spin resonance (ESR) spectra of spin labels have been used to monitor the melting transition in aminoacylated tRNA labeled at the a-amino group of the aminoacyl moiety (12) and in yeast tRNAPhe labeled in the penultimate, residue of the 3' terminus in which C-C-A has been replaced by C-s2C-A (13). In the current experiments, NMR spectra have been recorded of Esc...

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confidence: 99%

Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET.

Daniel¹,

Cohn²

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…Even when manganese (II) can substitute for magnesium (II), two different orders of catalytic efficiency have been observed. Figure 2 shows that these orders arise through the need for closer size-matching when the complexes are of the type enzyme-metal-substrate than when they are of the type enzyme-substrate-metal (38). In general, transition metals fail to activate magnesium enzymes as they bind the wrong groups of a protein-e.g., copper (II) and nickel (II) are often strong inhibitors (40).…”

Section: Probes For Group Iia Elementsmentioning

confidence: 99%

Biochemistry of Group IA and IIA Cations

Williams

1971

Advances in Chemistry

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The biological function of Group IA and IIA cations of the periodic table is reviewed against the background of their chemistry. Utilization of these cations arises from an ability to form different types of complex compounds, which is dependent upon the radius-ratio effect. If the details of their biochemistry are to be understood, new probe methods for following the cations in biological systems must be devised. Some possibilities based upon the principle of isomorphous replacement are described and tested.*"phe chemistry of the Group IA and IIA series of cations is relatively easy to understand as all its features are discernible from an inspection of the ionic model for chemical bonding (1, 2). This model shows that in an exchange reaction leading to a complex or an insoluble salt from a simple hydrate M(H20)n + L -> ML(H20)m + (n -m)H20 different cation stability sequences can be generated depending on the nature of the ligand, L, even though L is a simple anion or ligand. If L is a very large anion (usually of a strong acid) then the orders areand Ba 2 + > Sr 2 + > Ca 2 + > Mg 2 +which are sometimes called the lyotropic series. A clear-cut example is provided on association with sulfate anions either in solution or in the solid state but the orders are much more strikingly demonstrated in the solids where cooperative interactions are important. The opposite orders can be generated by simple small anions ( usually of necessity of a weak acid) such as hydroxide.

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“…The enzyme was first isolated from rabbit muscle (Kuby et al 1954) though it has been subsequently purified from a number of species (Watts 1973). The rabbit muscle enzyme, in particular, has undergone intensive investigation as the prototype of kinase-type reactions (Kuby and Noltmann 1962;Morrison and James 1965;Cohn 1970).…”

Section: Introductionmentioning

confidence: 99%

Some Properties of Human Skeletal Muscle Creatine Kinase

Cs¹,

Nicholson²,

O’Sullivan³

1977

Aust. Jnl. Of Bio. Sci.

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Creatine kinase has been purified from human skeletal muscle. The properties of the human enzyme are similar to those of the enzyme from rabbit muscle. The molecular weight was determined as approximately SO 000 with a probable two reactive sulphydryl groups per molecule. Manganous (II) ion was almost as effective as magnesium as the activating metal ion, and calcium and cobalt could also act in this capacity. Under standardized conditions the nucleotide specificity was ADP > dADP > IDP > GDP > UDP > XDP in the reverse reaction. No hydrolytic activity was observed with ATP.Initial velocity and product inhibition studies were used to determine various kinetic constants for the substrates of the enzyme. It was concluded that, as for the rabbit muscle enzyme, the reaction probably followed a rapid equilibrium random mechanism. Anomalous kinetic behaviour, however, was observed for the forward reaction for MgATp 2 -but not for creatine, when measurements were extended over a much wider range than normally used. The reciprocal plot of velocity as a function of substrate concentration gave a curve, concave downwards, instead of a straight line.

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Magnetic resonance studies of enzymesubstrate complexes with paramagnetic probes as illustrated by creatine kinase

Cited by 63 publications

References 46 publications

Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET.

Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET.

Biochemistry of Group IA and IIA Cations

Some Properties of Human Skeletal Muscle Creatine Kinase

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