David E. Metzler

Band Shapes of the Electronic Spectra of Complex Molecules

Structureless bands of ultraviolet absorption spectra of hydroxypyridine derivatives in solution have been examined. It is shown that the log–normal distribution provides a convenient four‐parameter empirical description of the bands and gives a much better fit than the two half‐width Gaussian. The log–normal yields a resolution of overlapping bands by a method of least squares that is very satisfactory. A simple configuration‐coordinate model is presented that lends some physical meaning to the parameters of the log–normal. It yields normalized third and fourth moments that are near those of the log–normal.

The oxidation of a reduced pyridine nucleotide analog by flavins

Abstract The kinetics of the non-enzymic hydrogen transfer from 1-propyl-1,4-dihydronicotinamide to riboflavin have been studied. The riboflavin anion does not react, but the cation formed by protonation of the isoalloxazine nucleus is estimated to react 10 4 times faster than neutral riboflavin. Oxidation rates with several other flavins are compared. Substituents on the ring nitrogen of the dihydronicotinamide which decrease the electron density of the ring cause a decrease in the oxidation rate. Oxidation is more rapid the more polar the solvent, and the higher the ionic strength. The rate of oxidation of the 4-deuterated 1-propyl-1,4-dihydronicotinamide is 3.2 times slower than that of the diprotiated form. Thus a hydrogen atom is removed in the rate limiting step of the oxidation. The results are consistent with oxidation by a hydride ion transfer mechanism.

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.

PHOTOCHEMICAL DEGRADATION OF FLAVINS‐IV. STUDIES OF THE ANAEROBIC PHOTOLYSIS OF RIBOFLAVIN*

Abstract A variety of substances which quench the fluorescence of riboflavin decrease the rate of anaerobic photobleaching (photolysis) of the flavin at concentrations which have little effect on the fluorescence. A semi‐quantitative estimate of the yield of various products of photolysis using thin layer chromatography and autoradiography with C14‐labelled riboflavin shows that at least ten radioactive products are formed. Whereas the yield of most of these is decreased by low concentrations of quenchers, such as phenol, some are decreased only at quencher concentrations high enough to decrease the fluorescence significantly. The effects of quenchers on the phosphorescence and ESR signal induced by light were also observed. It is concluded that lumichrome arises, in part, from degradation of the excited singlet state, but that 9‐formylmethylflavin and other products arise through the triplet state. A major product of anaerobic photolysis is a substance moving just ahead of riboflavin on chromatograms and which, like 9‐formylmethylflavin, is destroyed by treatment with sodium hydroxide. Effects of varying the nature of the solvent, the flavin structure and the pH are reported, and the kinetics of processes involved in the primary photochemical acts and in quenching are discussed.

Band-shape analysis and resolution of electronic spectra of pyridoxal phosphate and other 3-hydroxypyridine-4-aldehydes

The electronic absorption spectra of individual ionic forms of pyridoxal phosphate and of a series of related aldehydes have been evaluated together with pKa values. Spectral resolution with lognormal curves has permitted the quantitative description of equilibria for hydration and tautomerization. Precise values of peak positions for both aldehyde and hydrate forms have been obtained. Measurements of temperature-induced changes in the spectra have provided additional information. Knowing the hydration ratios and stepwide acid dissociation constants, it is possible to evaluate microscopic acid dissociation constants for both the aldehyde and hydrate forms of the compounds.

[77] Analyzing spectra of vitamin B6 derivatives

Publisher Summary This chapter describes methods for obtaining information about dissociation constants and various other chemical equilibria from spectral data. The methods are equally applicable to the study of many other light-absorbing substances. In this chapter, spectra are described by giving absorbances and wave numbers of peaks in kilokaysers (kK). This is desirable because of the direct proportionality of wave numbers with frequencies and energies. The 3-hydroxypyridine chromophore of vitamin B 6 is sensitively dependent upon structural factors and environment and provides a built-in indicator at the active sites of pyridoxal phosphate-dependent enzymes. Thus, the correct interpretation of subtle differences in the spectrum of the coenzyme may provide valuable information about events at the active site. This is one reason for the biochemists interest in spectra of the various forms of vitamin B 6 . The spectrophotometric determination of acid dissociation constants is simple and reliable for many aromatic substances.

2 Pyridoxal-Linked Elimination and Replacement Reactions

Publisher Summary The present biosynthetic pathways for amino acids involve the replacement of one β substituent by another through the action of pyridoxal phosphate (PLP)-containing enzymes. Likewise, in the catabolism of these amino acids, the first step is often the elimination of the β substituent. In a smaller number of cases there is substitution of an amino acid at the γ position; similarly, the removal of γ-substituent groups facilitates metabolism of such amino acids. All of these elimination and substitution reactions are catalyzed by PLP-requiring enzymes, and in the case of β elimination, nonenzymically by pyridoxal and pyridoxal analogs. The metabolic interrelationships involve 11 enzymes of this class. Both biosynthetic pathways, beginning with serine and aspartate, and catabolic reactions are demonstrated. These enzymes are especially numerous in sulfur metabolism. The first step in each β-elimination and replacement, and γ-elimination and replacement is the formation of an aldimine with PLP. The aldimine is presumably converted, by loss of the a-hydrogen of the amino acid to a quinonoid structure, which then eliminates the β substituent. The resulting unsaturated imine can either add a different nucleophile than the one eliminated or it can be converted to an α -imino acid and thence, in nonenzymic stage, to an α -keto acid and ammonia.

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.

[79] Pyridoxal 5′-phosphate and analogs as probes of coenzyme-protein interaction

Publisher Summary Pyridoxal phosphate (pyridoxal-P) provides a built-in indicator at the active sites of a substantial number of enzymes. Its absorption spectrum, which is often radically altered by substrates, quasisubstrates, or inhibitors, provides a means of directly monitoring intermediates in the catalytic process. A variety of analogs of the coenzyme is available and can often be substituted for pyridoxal-P to good advantage. Some analogs cause a covalent modification of enzymes. Pyridoxal phosphate and related compounds are widely used as modifying reagents for nonpyridoxal-P-dependent enzymes and other proteins. This chapter describes the sources of vitamin B 6 dependent enzymes; reversible removal of pyridoxal-P; reconstitution with analogs; analysis of absorption spectra, circular dichroism, and kinetics of reconstituted enzymes. Classical methods, such as salt fractionation, ion-exchange chromatography, and crystallization, are usually employed for the preparation of pyridoxal phosphate-dependent enzymes. Several techniques that seem to be particularly suitable for these enzymes are (1) heating in the presence of a competitive inhibitor to inactivate other proteins, (2) chromatography on hydroxyapatite, and (3) chromatography on carboxymethyl cellulose or carboxymethyl Sephadex.

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.