Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Å, enabling direct visualization of α-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i.e., insertion of peptides, ≈10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Å resolution. Hepatitis B virus capsids are icosahedral particles, ≈300 Å in diameter, made up of T-shaped dimers (subunit Mr, 16–21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and −7 (in the residual propeptide) in the “e-antigen” form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

Global air-sea flux of CO2: An estimate based on measurements of sea–air pCO2 difference

Approximately 250,000 measurements made for the pCO2 difference between surface water and the marine atmosphere, ΔpCO2, have been assembled for the global oceans. Observations made in the equatorial Pacific during El Nino events have been excluded from the data set. These observations are mapped on the global 4° × 5° grid for a single virtual calendar year (chosen arbitrarily to be 1990) representing a non-El Nino year. Monthly global distributions of ΔpCO2 have been constructed using an interpolation method based on a lateral advection–diffusion transport equation. The net flux of CO2 across the sea surface has been computed using ΔpCO2 distributions and CO2 gas transfer coefficients across sea surface. The annual net uptake flux of CO2 by the global oceans thus estimated ranges from 0.60 to 1.34 Gt-C⋅yr−1 depending on different formulations used for wind speed dependence on the gas transfer coefficient. These estimates are subject to an error of up to 75% resulting from the numerical interpolation method used to estimate the distribution of ΔpCO2 over the global oceans. Temperate and polar oceans of the both hemispheres are the major sinks for atmospheric CO2, whereas the equatorial oceans are the major sources for CO2. The Atlantic Ocean is the most important CO2 sink, providing about 60% of the global ocean uptake, while the Pacific Ocean is neutral because of its equatorial source flux being balanced by the sink flux of the temperate oceans. The Indian and Southern Oceans take up about 20% each.