Carol M. Metzler

Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate.

Two high resolution crystal structures of cytosolic aspartate aminotransferase from pig heart provide additional insights into the stereochemical mechanism for ligand-induced conformational changes in this enzyme. Structures of the homodimeric native structure and its complex with the substrate analog 2-methylaspartate have been refined, respectively, with 1.74-Å x-ray diffraction data to an R value of 0.170, and with 1.6-Å data to an R value of 0.173. In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligand-induced conformational change that involves a large movement of the small domain (residues 12–49 and 327–412) to produce a “closed” conformation. No such transition is observed in the other subunit (subunit 2), because crystal lattice contacts lock it in an “open” conformation like that adopted by subunit 1 in the absence of substrate. By comparing the open and closed forms of cAspAT, we propose a stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (φ,ψ) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix. The calculated free energy for the burial of this “hydrophobic plug” appears to be sufficient to serve as the driving force for domain closure.

The Widespread Applicability of Lognormal Curves for the Description of Absorption Spectra

We have fitted over 160 electronic absorption spectra of over 95 aromatic compounds and some other substances with lognormal distribution curves to evaluate the shapes of the bands. The compounds include simple benzene derivatives, phenols, anilines, benzaldehyde, benzoic acid, nitrobenzene, and potassium permanganate. The well-defined n-π* transitions of acetone, benzaldehyde, nitrobenzene, and pyrazine are included. Effects of solvents have been studied in a number of cases. The fitting of absorption bands with lognormal curves is compared with resolution with Gaussian curves or with other types of skewed Gaussian curves. We conclude that the lognormal curve provides an excellent approximation to the shapes of spectral bands in aromatic as well as many other organic and inorganic compounds. The ability to analyze complex spectra by resolution with lognormal curves may be usefully applied in a number of ways.

Analogs of Glutamic Acid: Synthesis and Biological Evaluation

Abstract The reaction of bromodiester 6 with base in liquid ammonia affords good yields of aminocyclopropane diesters 8 and 9. The corresponding diacids were not inhibitors of AAT.

NMR spectra of exchangeable protons of pyridoxal phosphate-dependent enzymes

We have recorded 1H NMR spectra in H2O for exchangeable protons of four pyridoxal phosphate-dependent enzymes: D-serine dehydratase, aspartate aminotransferase, tryptophan: indole-lyase and glutamate decarboxylase. The molecular masses range from 48-250 kDa. In every case there are downfield peaks which are lost when the apoenzyme is formed. In most cases some peaks shift in response to interactions with substrates and inhibitors and with changes in pH. We associate one downfield resonance with the proton on the ring nitrogen of the coenzyme and others with imidazole groups that interact with coenzyme or substrates. The chemical shift for the coenzyme-bound proton differs for free enzyme, substrate Schiff base or quinonoid forms.

[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.

Studies on the Inactivated Coenzyme in the Beta Subform of Cytosolic Aspartate Aminotransferase

A major product released by heat from the 340 nm chromophore of the beta subform of cytosolic aspartate aminotransferase has been identified conclusively as pyridoxamine 5′-phosphate. The N-phosphopyridoxyl derivative of 5-oxoproline has been identified as a minor product. We suggest that the beta chromophore is a stabilized ketimine that can isomerize to an aldimine and release, in part, pyridoxal phosphate at high pH. It may have arisen from a reactive 5-carbon substrate.

1H and15N NMR Spectroscopy of Aspartate Aminotransferase and Related Schiff Bases and Tautomerism in Enzyme Active Sites

Downfield resonances from hydrogen bonded protons are delicate sensors of changes in charge distribution in active sites. They indicate that a tight electrostatic interaction of the phenolate oxygen with the charged amino group of substrate, coenzyme, or catalytic lysine is maintained in the various enzyme-substrate complexes which can be viewed as a set of interconverting tautomers. The resonance of the Schiff base N-H proton was observed in nonenzymatic Schiff bases of PLP with amino acids in 90%H2O/10%D2O at ~15 ppm.