Cadmium-113 NMR of carbonic anhydrases: effect of pH, bicarbonate, and cyanide.

Abstract

ABSTRACT"13Cd-Substituted human and bovine erythrocyte carbonic anhydrases have been studied by "13Cd NMR as a function of pH and bicarbonate concentration. Plots of chemical shift versus pH give sigmoidal titration curves in the pH range of the study, 6.9 to 10.5. The pKa values vary from 9.2 to 9.7, which correlates well with available activity profiles for the Cd-enzymes. Because the samples contain no buffers and no anions other than hydroxide, the results point to the existence of high and low pH forms of the enzymes in rapid exchange and differing in inner sphere coordination. When bicarbonate is added to the samples, upfield shifts are produced which eventually level off. Only a single CN-binds to the metal for all three enzymes. These observations are best explained by a rapid exchange among three species in which the open coordination site of the metal ion is occupied by hydroxide, water, or bicarbonate, as in the scheme: EOH-;± E-H20 ± E-HCQ3. Carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1) are zinc metalloenzymes that are found in animals, plants, and certain bacteria and catalyze the reversible hydration of carbon dioxide (CO2 + H20 :=± HCO% + H+), the hydrolysis of certain esters, and various other reactions (1). Human carbonic anhydrase B (hCAaseB) and C (hCAaseC) and bovine carbonic anhydrase (bCAase) from human and bovine erythrocytes are monomeric enzymes of molecular weight k29,000, each molecule containing a single equivalent of firmly bound Zn(II) which is required for catalytic activity. The Zn can be replaced by various divalent metal cations, including Co(II) and Cd(II) (2). The activity appears to be controlled by a single titratable group with a pKa value around 7; the high pH form of the enzyme is required in the hydration reaction and the low pH form is required in the dehydration. A parallel between the spectral changes of Co(II)-enzymes and their activities versus pH has been taken as support for a mechanism in which the low and high pH forms are zinc-coordinated water and hydroxide ion, respectively (3, 4). However, the identity of the catalytic group remains in dispute, and alternative mechanisms have been proposed (5).In this report we present a systematic 1"3Cd NMR study of hCAaseB, hCAaseC, and bCAase as a function of pH and bicarbonate concentration. Such 113Cd NMR studies of metalloproteins have been shown to provide information regarding the coordination sphere of the metal ion (6-10).MATERIAL AND METHODS Enzymes. hCAaseB, hCAaseC, and bCAase were prepared by affinity chromatography according to the Khalifah et al. (11) modification of the method of Osborne and Tashian (12), followed by chromatography on DEAE-cellulose (Whatman DE-23). Enzyme concentrations were estimated spectrophotometrically by using the following extinction coefficients (Ag9o) and molecular weights: 18.7 cm-1, 16.3 cm-1, and 19.0 cm-1 and 29,300, 28,850, and 29,500 for hCAaseC, hCAaseB, and bCAase, respectively (1, 13,14). The metal-free enzymes were prepared by dialysis against 75 mM pyridine-2,4...

Synthesis, structure, and platinum-195 NMR solution studies of a reactive binuclear platinum(III) complex: bis(ethylenediamine)nitratonitrobis(2-pyridonato)diplatinum(2+) dinitrate hemihydrate

“…For typical procedures used in our laboratory see ref 12. 195Pt NMR spectra were acquired on a Bruker WM-250 spectrometer operating at 53. 6 MHz. The 90°p ulse in a fixed-frequency, 10-mm probe was 25 gs.…”

Pentagonal-bipyramid geometry and cadmium-113 NMR chemical shifts: crystal structures and solution and solid-state cadmium-113 NMR of bis(.mu.-o-hydroxybenzoato)bis(o-hydroxybenzoato)tetraaquodicadmium(II) and tris(pyridine)bis(o-hydroxybenzoato)cadmium(II)

“…113Cd NMR with a chemical shift range of ~900 ppm has been shown to be a sensitive probe of metal ion sites in a wide variety of compounds, ranging from organometallic to bio- 0= 96.23 (3)°pobsd= 1.83 (2)g/cm3 Scaled = 1-86 g/cm3 Z = 4 fw = 422.66 ( ) = 0.710 73 A graphite monochromator used; 26 = 6.1°s pace group P2Ja hOl, h = 2n + 1; OkO, k = 2n + 1 absent size of crystal: 0.20 X 0.20 X 0.50 mm µ = 14.8 cm'1 faces of the form {001}, {010}, {100}, [101], [101] abs cor made; max/min transmissn factors 0.765/0.485 P factor = 0.030 in a(F02) = [o(/taw)2 + (Plxaw)2 V'VlP and w = l/a(FQ)2 data considered nonzero if F2 > 4o(F2) 6614 independent hkl's measd in -2 mode to 26 = 70°5 058 reflcns used to solve and refine structure variable scan speed with preliminary scan speed of 4°/min in 20 25 reflcns used in orientation matrix (checked every 24 h) 3 std reflcns monitored every 100 reflcns; decay less than 2% I room temp ~18 °C structure refined by full-matrix least squares, including anisotropic temperature factors and anomalous dispersion corrections with weights based upon intensity statistics; function minimized = s¿w¡[ IF0¡I-IFC¡-I]2 largest shift at end of refinement: 0.55 final least squares performed on Amdahl V6 (f s from ref 41) no. of variables: 208 final R = 0.028, weighted R = 0.042 error of observn of unit wt: 1.27 (b) Tris(pyridine)bis(ohydroxybenzoato)cadmium(II) a = 10.187 (4) A b = 14.846 (5) A c = 17.965 (5) A 0= 93.79 (3)°p obsd= 1.54 (2)g/cm3 pcalcd = 1.53 g/cm3 Z = 4 fw= 623.9 ( ) = 0.710 73 A graphite monochromator used; 26 = 6.1°s pace group P2Jn hOl, h + l = 2n + 1; OkO, k = 2n + 1 absent size of crystal: 0.20 X 0.25 X 0.30 mm µ = 8.5 cm"1 faces of the form {001}, {100}, {011} abs cor made; max/min transmissn factors 0.855/0.789 P factor = 0.030 in o(F02) = [o(/raw)2 + (^raw)2]1'2/^a nd w= 1 /a(F0)2 data considered nonzero if F2 > 4a{F2) 8171 independent hkl's measd in cu-26 mode 4944 reflcns used to solve and refine structure variable scan speed with preliminary scan speed of 4°/min in 26 25 reflcns used in orientation matrix (checked every 24 h) 3 std reflcns monitored every 100 reflcns; decay less than 2% / room temp ~18 °C structure refined by full-matrix least squares, including anisotropic temp factors and anomalous dispersion corrections with weights based upon intensity statistics; function minimized = SiWiMFoil-IFeil]2 largest shift at end of refinement; 1.57o final least squares performed on Amdahl V6 (fs from ref 41) no.…”

Cadmium-113 and carbon-13 nuclear magnetic resonance spectrometry of cadmium-amino acid complexes

“…Increased sampling speeds have instead been obtained by bubble-gating techniques (3)(4)(5) and by the development of various flow-injection analysis (FIA) systems (6)(7)(8)(9)(10). FIA operates under non-steady-state conditions and gives sharp sample peak readout, as reviewed by several authors (6-10).…”