Neil E. MacKenzie

A carbon-13 nuclear magnetic resonance analysis of the products of glucose metabolism in Leishmania pifanoi amastigotes and promastigotes.

The major products of glucose metabolism were determined for amastigotes and promastigotes of Leishmania (mexicana) pifanoi under aerobic and anaerobic conditions using carbon-13 nuclear magnetic resonance. Under aerobic conditions, the major products for both forms were carbon dioxide, succinate, malate, acetate and alanine. Succinate was the dominant metabolite of promastigotes, whereas acetate and alanine were most abundant with amastigotes. Under anaerobic conditions, promastigotes produced glycerol as the dominant metabolite, along with lesser amounts of succinate, acetate and alanine; acetate and alanine remained major metabolites in amastigotes, with an increase in the relative amount of succinate and the production of some glycerol. Promastigotes generated carbon dioxide at a 5-fold greater rate than amastigotes under aerobic conditions, but this rate was reduced by more than 95% in the absence of oxygen. Amastigotes were relatively less affected by lack of oxygen and produced carbon dioxide at a rate comparable to promastigotes under anaerobic conditions. The presence of carbohydrates with a possible role in storage was detected in both promastigotes and amastigotes.

Pro→Ala-35 Rhodobacter capsulatus cytochrome c2 shows dynamic not structural differences : a 1H and 15N NMR study

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild‐type and mutant Pro → Ala‐35 Rhodobacter capsulatus cytochrome c 2 are indistinguishable. The ring resonances of Phe‐51 and Tyr‐53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly‐34 and the NπH of His‐17 increase by ≈ 102 in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3 αNH. 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.

Uniformly 13C-enriched substrates as n.m.r probes for metabolic events in vivo. Application of double quantum coherence to a biochemical problem

Glycolysis in Escherichia coli has been monitored in vivo by 13C n.m.r. spectroscopy and the biochemical pathways involved in succinate biosynthesis have been evaluated by analysis of the complex resonance signals, using double quantum coherence.

13C-NMR analysis of alanine metabolism by isolated perfused livers from C3HeB/FeJ mice infected with African trypanosomes.

1. Isolated perfused livers from mice infected with Trypanosoma brucei rhodesiense formed substantially more [3-13C]-lactate from [3-13C]-alanine than livers from uninfected mice. Quantities formed by infected livers increased as infection progressed. 2. Infected livers produced more 13C-labeled glutamate and glutamine, with label scrambled between C-2 and C-3. Scrambling also produced [2,3-13C]-aspartate, [2-13C]-alanine and [2-13C]-lactate. Delayed appearance of label in C-4 of glutamate/glutamine in infected livers reflects significant endogenous stores of unlabeled acetyl CoA. 3. Although differences do exist in catabolism of [3-13C]-alanine by perfused livers from infected and control mice, trypanosomiasis does not cause permanent breakdown or blockage of hepatic alanine metabolism.

Amide hydrogen exchange of the central B-chain helix within the T- and R-states of insulin hexamers

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

Biosynthesis of the antitumour catharanthus alkaloids: the fate of the 21′α-hydrogen of anhydrovinblastine

[21′α-3H,methyl-14C] Anhydrovinblastine is incorporated into vinblastine by cell-free preparation of Catharanthus roseus without loss of 3H.

Formation of grindelane dimers by microbial transformation

Abstract The material balance for the biotransformation of grindelic acid by Aspergillus niger was determined along with the isolation of two novel dimers identified as methyl-8α-hydroxy-grindelate-7β- O -7′β-ether hydrate and methyl-3β-acetoxy-8α-hydroxy-grindelate-7β- O -7′β-ether hydrate. Also identified were the known 19-hydroxy-grindelic acid and the novel 6α,17-dihydroxy-grindelic acid. All compounds were isolated as their methyl ester acetate derivatives. The known 3β-hydroxy-7α,8α-epoxy grindelic acid was the sole product formed when 3β-hydroxy-grindelic acid was used as the substrate for biotransformation.

Proton nuclear magnetic resonance study of the histidine residues of pituitary bovine growth hormone

The pKa value of histidines 20, 22 and 170 of pituitary bovine growth hormone (pbGH) and their C2H deuterium exchange rates have been determined by high-resolution 1H-NMR spectroscopy. Partial assignment of these parameters indicate that His-170 is a surface residue with a half-life for deuterium exchange at the C2H position of approx. 36 h and a pKa value of 5.68 +/- 0.11. A reversible conformational change of pbGH has also been characterized in terms of the involvement of either histidines 20 or 22. At or near physiological pH values, one of these residues is expelled from a buried, hydrophobic position to become fully solvent-exposed. The disparity in pKa values between histidines 20 and 22 (4.67 +/- 0.20 and 5.94 +/- 0.10, in some combination) has been shown by computer modeling, to be compatible with these residues residing in an alpha-helical region of the protein.

N.m.r. studies of enzyme mechanism. Comparison of the crystal structure and solid state 13C and 15N n.m.r. spectra of a carboxypeptidase A complex with glycyl tyrosine

It is shown that solid state n.m.r. spectroscopy can be used to determine the extent of cleavage of the scissile amide bond in glycyl tyrosine, a slow substrate for the enzyme carboxypeptidase A in the crystalline enzyme substrate complex.

Pro → Ala-35Rhodobacter capsulatuscytochromec2shows dynamic not structural differences: A1H and15N NMR study

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild‐type and mutant Pro → Ala‐35 Rhodobacter capsulatus cytochrome c 2 are indistinguishable. The ring resonances of Phe‐51 and Tyr‐53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly‐34 and the NπH of His‐17 increase by ≈ 102 in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3 αNH. 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.