Distance determinations between the metal ion sites of Escherichia coli glutamine synthetase by electron paramagnetic resonance using chromium(III)-nucleotides as paramagnetic substrate analogs

Abstract

The substitution-inert Cr(III)-nucleotides, CrADP and CrATP, were tested as inhibitors of unadenylylated Escherichia coli glutamine synthetase. Both compounds were linear competitive inhibitors vs. MgATP in the biosynthetic assay which consists of following the formation of glutamine from glutamate, ATP and ammonia. The K\ values were 9.6 ± 0.6 µ and 25 ± 1 µ for CrATP and CrADP, respectively. The paramagnetic property of the Cr(III)-nucleotides (S = 3/2) was used to study the interaction between Mn(II) {S = 5/2) bound at the n \ "tight" metal ion site and Cr(III)-nucleotide bound at the «2 metal ion site. Addition of a saturating amount of CrATP produces a 60% decrease in the electron paramagnetic resonance spectrum (EPR) of enzyme-bound Mn(II). This electronic spin-spin interaction between Mn(II) and Cr(III) was analyzed at both 9 and 35 GHz using the J. S. Leigh theory ((1970) J. Chem. Phys. 52, 2608) of dipolar electronic relaxation. Titration experiments with CrATP were conducted by following the decrease in the EPR spectral amplitude of enzyme-Mn(II) and a £D value of 0.30 ± 0.04 mM was calculated. A distance of 7.1 Á between Mn and Cr was obtained by analysis of these EPR data using (jlutamine synthetase from Escherichia coli is composed of 12 identical subunits and is known to catalyze several reactions in addition to the biosynthesis of glutamine from glutamate, ammonia, and ATP (Stadtman & Ginsburg, 1974). The enzyme shows an absolute requirement for two divalent cations per subunit for catalysis to occur (Hunt et al., 1975).The unadenylylated enzyme binds Mn(II) at these two sites with affinities that differ by approximately two orders of magnitude in the absence of substrates (Denton & Ginsburg, 1969;Hunt et al., 1975;Villafranca et al., 1976;Shrake et al., 1977). The first ("tight") metal ion site is known to produce conformational changes in the protein but may also be near the catalytic site (Villafranca et al., 1976). Hunt et al. (1975) demonstrated that the second metal ion site is the metal nucleotide site.Recently, Cleland and co-workers have developed procedures for the synthesis and purification of substitution-inert Cr(III)-nucleotide complexes and have used them as dead-end inhibitors to study the kinetic mechanisms of several kinases (DePamphilis & Cleland, 1973; Janson & Cleland, 1974a,b;Danenberg & Cleland, 1975). Also, the paramagnetism and nonlability of the Cr(III)-nucleotide complexes make them useful for NMR experiments. Such studies have been con-* From the

Manganese(II) as magnetic relaxation probe in the study of biomechanisms and of biomacromolecules

“…From the effect of adding Cr(III) or Co(II) to bound Mn(II), a dual divalent cation requirement was assayed for some enzymes. 155,179,180,182,183 The probe to probe distance was shown to depend on the presence of substrates and inhibitors,182 allowing a direct proof of induced conformational changes.…”

Homopolygalacturonan Molecular Size in Plant Cell Wall Matrices Via Paramagnetic Ion and Nitroxyl Amide Dipolar Spin-Spin Interactions

“…Generally, dipolar interactions (10) are one of the most useful homogeneous interactions in electron paramagnetic resonance spectroscopy. These spin-spin interactions have long been utilized in molecular biology (11)(12)(13)(14)(15) to determine, for example, metal-metal distances between two binding sites on the surface of an enzyme (13). In previous studies (16)(17)(18) direct evidence was presented that certain paramagnetic species (Mn2", Cu2+, or a nitroxyl amine) can ionically, as a metal complex, or covalently, as an amide, bond to plant homopolygalacturonans in a spatially sequential fashion.…”

Probing the catalytic roles of n2‐site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis

“…The metal ion at the n, site was suggested to be involved in glutamate binding (Villafranca et al, 1975;Balakrishnan & Villafranca, 1978;Hunt & Ginsburg, 1980) and may further function as an electrophilic catalyst in polarizing the carbonyl group of the covalent intermediate, y-glutamyl phosphate, for attack by ammonia. Additional support for the role of the n, metal ion in catalysis comes from EPR studies involving the transitionstate analog methionine sulfoxime (Eads et al, 1988).…”