Paul H. Rogers

Preliminary crystallographic study of aspartate: 2-oxoglutarate aminotransferase from pig heart*

Well-ordered crystals of aspartate: 2-oxoglutarate aminotransferase have been grown by vapor diffusion from solutions of polyethylene glycol. X-ray diffraction patterns show that they belong to the orthorhombic space group P212121 with unit cell dimensions a = 124·7 , b = 130·9 , and c = 55·7 . The asymmetric unit consists of one dimer of molecular weight 92,688. The diffraction pattern extends beyond 2·8 , indicating that this crystal form is suitable for high resolution X-ray analysis.

X-ray diffraction studies of a partially liganded hemoglobin, [α(FeII-CO)β(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [α(FeII-CO)β(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3·0 A, shows that (1) the Mn(II)-substituted β subunits are structurally isomorphous with normal deoxy β subunits, and (2) CO binding to the α subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the α heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the α1β2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.

High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini.

The crystal structures of three mutant hemoglobins reconstituted from recombinant beta chains and authentic human alpha chains have been determined in the deoxy state at 1.8-A resolution. The primary structures of the mutant hemoglobins differ at the beta-chain amino terminus. One mutant, beta Met, is characterized by the addition of a methionine at the amino terminus. The other two hemoglobins are characterized by substitution of Val 1 beta with either a methionine, beta V1M, or an alanine, beta V1A. All the mutation-induced structural perturbations are small intrasubunit changes that are localized to the immediate vicinity of the beta-chain amino terminus. In the beta Met and beta V1A mutants, the mobility of the beta-chain amino terminus increases and the electron density of an associated inorganic anion is decreased. In contrast, the beta-chain amino terminus of the beta V1M mutant becomes less mobile, and the inorganic anion binds with increased affinity. These structural differences can be correlated with functional data for the mutant hemoglobins [Doyle, M. L., Lew, G., DeYoung, A., Kwiatkowski, L., Noble, R. W., & Ackers, G. K. (1992) Biochemistry preceding paper is this issue] as well as with the properties of ruminant hemoglobins and a mechanism [Perutz, M., & Imai, K. (1980) J. Mol. Biol. 136, 183-191] that relates the intrasubunit interactions of the beta-chain amino terminus to changes in oxygen affinity. Since the structures of the mutant deoxyhemoglobins show only subtle differences from the structure of deoxyhemoglobin A, it is concluded that any of the three hemoglobins could probably function as a surrogate for hemoglobin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Site‐directed mutations of human hemoglobin at residue 35β: A residue at the intersection of the α1β1, α1β2, and α1α2 interfaces

Because Tyr35β is located at the convergence of the α1β1, α1β2, and α1α2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation‐induced changes in functional and structural properties are found for the recombinant hemoglobins βY35F and βY35A. Oxygen equilibrium‐binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35β results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen‐binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in βY35F and increases in βY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in βY35F or βY35A. X‐ray crystal structures of deoxy βY35F and βY35A are highly isomorphous with the structure of wild‐type deoxyhemoglobin. The βY35F mutation repositions the carboxyl group of Asp126α1 so that it may form a more favorable interaction with the guanidinium group of Arg141α2. The βY35A mutation results in increased mobility of the Arg141α side chain, implying that the interactions between Asp126α1 and Arg141α2 are weakened. Therefore, the changes in the functional properties of these 35β mutants appear to correlate with subtle structural differences at the C terminus of the α‐subunit.

[80] Investigation of crystalline enzyme—Substrate complexes of pyridoxal phosphate-dependent enzymes☆

Publisher Summary Many pyridoxal phosphate (pyridoxal-P) dependent enzymes have been crystallized, but few of the crystals have been studied either by X-ray crystallography or by other physical techniques. Recently, three groups have initiated crystallographic studies on aspartate aminotransferases. The cytosolic enzyme from chicken hearts and from pig hearts has been prepared in orthorhombic forms, while the mitochondrial isoenzyme of chicken heart has been crystallized in a triclinic form. Many pyridoxal-P enzymes form very small and relatively insoluble crystals. However, if the solubility of these enzymes is increased by changing the pH or ionic composition of the buffer, it may be possible to use the polyethyleneglycol method to obtain larger crystals. This chapter describes the preparation of crystals of the cytosolic isoenzyme of aspartate aminotransferase from pig heart and crystals of enzyme-substrate or enzyme-inhibitor complexes. Crystals of enzyme containing analogs of pyridoxal-P can be prepared by reconstituting the apoenzyme and then treating it as above for the native enzyme.

X-Ray Crystallographic, 19F-NMR and UV-Visible Spectroscopic Studies of Pig Heart Cytosolic Aspartate Aminotransferase

Structures determined by x-ray diffraction have been refined at 2.4–2.6A for the native enzyme, the external aldimine formed with α-methylaspartate and the ketimine species formed with L-glutamate. These results have been correlated with those of microspectrophotometry on crystals and are discussed in relationship to UV-visible spectra and 19F NMR spectra in solut ions.