Lawrence C. Kuo

Determination of the Zero-Field Splitting Constants of High-Spin Metalloproteins by a Continuous-Wave Microwave Saturation Technique

Abstract A method is described for determining the zero-field splitting energy of high-spin metalloenzymes and proteins by a continuous-wave saturation technique with a conventional EPR spectrometer. The method requires analysis of graphical estimates of P 1 2 from saturation curves as a function of temperature. Under the condition that the signal amplitude depends only on the spin-lattice relaxation probability, the temperature dependence of P 1 2 provides an estimate of the zero-field splitting energy for spin-lattice relaxation via an Orbach process. The zero-field splitting energies of metmyoglobin and metmyoglobin fluoride are estimated at 22 and 12 cm−1, respectively, in excellent agreement with published results obtained by magnetic susceptibility and pulse saturation recovery methods. The zero-field splitting of methemoglobin is sensitive to the binding of inositol hexaphosphate. This change is shown to be consistent with the change in iron-porphyrin stereochemistry induced by binding of the organic phosphate molecule to the protein. The method can be applied to a wide variety of high-spin paramagnetic systems.

Catalytic conformation of carboxypeptidase A. The structure of a true reaction intermediate stabilized at subzero temperatures.

Abstract The structure of the mixed anhydride, acyl-enzyme intermediate of the esterolytic reaction of carboxypeptidase A is characterized by application of cryoenzymologic, magnetic resonance, and molecular graphics methods with use of the Co2+-substituted enzyme and the specific spin-label ester substrate O-3-(2,2,5,5-tetra-methylpyrrolinyl-1-oxyl)-propen-2-oyl- l -β-phenyllactate. A radial separation of 7·7 A between the active site Co2+ and the nitroxide group in the low temperature-stabilized acyl-enzyme intermediate is determined on the basis of their spin-spin (dipole-dipole) interactions. Application of molecular graphics techniques shows that the only configuration of the substrate that is sterically accommodated by the active site yields a calculated metal ion-to-nitroxide distance of 7·8 A. Steric accommodation of the spin-label in the active site requires severe torsional distortion around the aliphatic double bond of the propenoyl side-chain. Examination of the structure of the enzyme: spin-label intermediate reveals that the distortion arises from steric interactions of the pyrrolinyl group with the protein at a position that corresponds to the site occupied by the penultimate amide residue of an oligopeptide substrate from the site of cleavage. Together with kinetic data showing that hydrolysis of the spin-label is governed by rate-limiting deacylation, the results indicate that geometric distortion of substrates by secondary interactions with the enzyme, in general, is an obligatory part of the catalytic action of carboxypeptidase A. When viewed with respect to requirements for stereoelectronic control of bond cleavage in tetrahedral adducts of esters and amides (Deslongchamps, 1975) the results suggest that torsional distortion during catalysis results in rotation around the scissile bond of the substrate, and that this rotation is required to form the mixed anhydride reaction intermediate. These findings further support the interpretation that the hydrolysis of esters and amides catalyzed by carboxypeptidase A proceeds according to similar mechanisms except that formation of the mixed anhydride is rate-determining in peptide hydrolysis while deacylation of the mixed anhydride is rate-limiting in ester hydrolysis. Additionally, in this study application of the extension of the theory of the Solomon-Bloembergen-Morgan equations derived by Lindner (1965) for paramagnetic metal ions with S ≥ 1 demonstrates that the zero-field splitting of the high-spin Co2+ in the metal-substituted enzyme has no significant influence in determination of the relaxation enhancement of solvent protons by the active site metal ion.

The catalytic action of Zn2+-metalloenzymes requires a penta-coordinate metal-OH2 complex

A review is presented of the spectroscopic and chemical evidence that the tetra-coordinate active site metal ion of carboxypeptidase A and liver alcohol dehydrogenase accommodates both the substrate and a water molecule in catalytically competent reaction intermediates to form a penta-coordinate complex. In carboxypeptidase A ionization of the metal-OH2 complex forms a potent metal-hydroxide nucleophile for breakdown of the acylenzyme (mixed anhydride) reaction intermediate in esterolysis, while in liver alcohol dehydrogenase a neutral metal-OH2 species serves as a conduit for proton abstraction from the alcoholic hydroxyl group. These results together with correlated observations that the catalytic activity of other Zn2+-metallo-enzymes depends on ionization of a Zn2+-OH2 complex suggests that a penta-coordinate metal-OH2 species is an obligatory species in the catalytic action of this class of enzymes.