Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I.

This research investigates the properties of horse carbonic anhydrase I enzyme using proton nuclear magnetic resonance, identifying several key properties and changes in the enzyme structure compared to its human equivalent, showing specific differences in the histidine residues.

Overview of the experimental setup

• The researchers executed a nuclear magnetic resonance (NMR) study at 250 MHz on horse carbonic anhydrase I, a type of enzyme found in horses and similar to a similar one found in humans.
• These tests were carried out under diverse conditions, changing the pH to observe how this affects the enzyme.

Findings related to Histidine residues

• The tests successfully identified specific resonances, or nuclear spin transitions, related to the histidine C-2 and C-4 protons, an amino acid found within the enzyme. Eight resonances were found corresponding to C-2 protons, and four corresponding to C-4 protons.
• These resonances were then assigned to individual histidine residues within the enzyme molecule.

Comparison between Horse and Human carbonic anhydrases I

• A comparison was made between horse and human carbonic anhydrases I, showing significant similarities between both forms.
• Observe that the human enzyme has three titratable histidine residues in its active site, whereas the horse enzyme only has two. The histidine residue His-67 found in human enzymes is replaced by glutamine (Gln) in the horse enzyme, representing a key difference between both species.

Effects of the substitution

• Substituting His-67 with glutamine in the horse enzyme does have slight, but significant effects on the other active site histidines, His-64 and His-200.
• The researchers also noted that His-64 in horse isoenzyme I has a notably low pKa value, agreeing with previous observations in human isoenzyme I. The pKa value corresponds to the pH at which half the enzyme molecules are in their protonated form.