Biomedical subjects

D G Lambright

Publications and source records attributed to D G Lambright.

22 records · Page 2Linked to original sources

PubMed1989

Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively conservative change of a surface residue can substantially perturb ligand and proton exchange dynamics in a manner that is not readily predicted from the static structures.

Amino Acids↗

PubMed1989

Electrostatic interactions in wild-type and mutant recombinant human myoglobins.

Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pKa of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior.

Codon↗

PubMed1985

Lactose synthase: effect of alpha-lactalbumin on substrate activity of N-acylglucosamines.

N-Acetyl-, N-propionyl-, N-butyryl- and N-valerylglucosamines were synthesized as topographical probes to localize further the interaction site of alpha-lactalbumin on galactosyltransferase. All these compounds were found to be substrates for galactosyltransferase with Km values in the millimolar range. In the presence of alpha-lactalbumin, the Michaelis-Menten constants were diminished. However, the effect on the initial rates of these reactions varied. Thus, at low N-acylglucosamine concentrations, alpha-lactalbumin activated the enzyme activity, but at high concentrations, alpha-lactalbumin became inhibitory. This mixed-type inhibition kinetics indicated that a quaternary complex between galactosyltransferase, alpha-lactalbumin, Mn2+-UDPgalactose and N-acylglucosamine existed during the catalytic process. The ability of these N-acylglucosamine substrates to bind to lactose synthase complex was further substantiated by the physical association of galactosyltransferase onto the solid-bound alpha-lactalbumin in the presence of any one of these compounds. The data revealed that the presence of the N-acyl group up to five carbons in length did not interfere with the interaction between alpha-lactalbumin and galactosyltransferase, suggesting that alpha-lactalbumin was not bound in the vicinity of the C-2 region of the monosaccharide site. The inhibitory effect of alpha-lactalbumin on N-acyllactosamine formation is probably a consequence of conformational changes of galactosyltransferase.

Animals↗

PubMed1985

Association-dissociation modulation of enzyme activity: case of lactose synthase.

Lactose synthase was found to show anomeric preference for beta-D-glucose. This information was utilized in the design of methyl, ethyl, propyl, butyl, and pentyl N-acetyl-beta-D-glucosaminides, which were subsequently demonstrated to be substrates for galactosyltransferase with apparent Km values in the low millimolar range. alpha-Lactalbumin competitively inhibits the transferase activity against these N-acetylglucosamine derivatives. This pattern of inhibition has also been observed when the dimer, trimer, and tetramer of N-acetylglucosamine and ovomucoid served as the galactose acceptor. The data suggest that the binding of alpha-lactalbumin and the N-acetylglucosamine derivatives is mutually exclusive. This assertion is further supported by the inability of methyl and butyl N-acetyl-beta-D-glucosaminides to facilitate retention of galactosyltransferase on a column of alpha-lactalbumin immobilized onto Sepharose. Free N-acetylglucosamine, on the other hand, does cause retention of the transferase under the same conditions. Thus, alpha-lactalbumin must bind to a region on galactosyltransferase in close proximity to the monosaccharide binding site and exert its substrate-specifying action through competitive and mutually exclusive binding with the N-acetylglucosamine analogues accompanied by an increased affinity for glucose. In short, our substrate analogue studies have revealed that the association-dissociation modulation of galactosyltransferase activity is effected through a topographical blockade of glycoprotein binding by alpha-lactalbumin.

Animals↗