Structural analysis of zinc substitutions in the active site of thermolysin.

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.     

Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy.

Far-UV CD, 1H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 micrograms total protein) and, unlike either CD or 1H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.     

Effects of Na readmission on cellular45Ca fluxes in Na-Depleted guinea pig taenia coli

Summary The removal of Na from the medium causes a cellular Ca uptake in the smooth muscle of the guinea pig taenia coli which is rapidly reversed if medium Na is readmitted. This net extrusion was characterized in tissues which were first Na-depleted in a zero-Na (sucrose) solution. Li was able to substitute for Na in mediating this effect. K was also able to mimic Na in this respect if the depolarization-mediated Ca influx caused by the isotonic K solution was blocked with 10^–5 m D-600. The net Ca extrusion upon Na readmission was due to a small decrease in Ca influx, as well as a marked increase in the transmembrane Ca efflux rate, as revealed by⁴⁵Ca washout experiments. The increased⁴⁵Ca efflux upon Na readmission could be mimicked by Li, K, choline and tris. We conclude that the Na/Ca-exchange hypothesis is insufficient to explain these data, in that both Ca extrusion and⁴⁵Ca efflux can be stimulated in the absence of a Na gradient, or in the absence of any monovalent cationic gradient. These observations are discussed in terms of a possible intracellular competition of Ca and monovalent cations for anionic binding sites, as well as with regard to a possible direct stimulation of a plasmalemmal CaATPase by monovalent cations.     

A vibrational study of the CD2 stretching bands of selectively deuterated palmitic and stearic acids

The C-D stretching regions of the infrared and Raman spectra of 14 different palmitic and stearic acids containing isolated CD₂ groups are reported. Anomalous behaviour is observed when substitution occurs near the terminal methyl group, which behaviour can not be explained in terms of crystal field effects.     

Molecular considerations in the evolution of bacterial genes

Summary Synonymous and nonsynonymous substitution rates at the loci encoding glyceraldehyde-3-phosphate dehydrogenase (gap) and outer membrane protein 3A (ompA) were examined in 12 species of enteric bacteria. By examining homologous sequences in species of varying degrees of relatedness and of known phylogenetic relationships, we analyzed the patterns of synonymous and nonsynonymous substitutions within and among these genes. Although both loci accumulate synonymous substitutions at reduced rates due to codon usage bias, portions of thegap andompA reading frames show significant deviation in synonymous substitution rates not attributable to local codon bias. A paucity of synonymous substitutions in portions of theompA gene may reflect selection for a novel mRNA secondary structure. In addition, these studies allow comparisons of homologous protein-coding sequences (gap) in plants, animals, and bacteria, revealing differences in evolutionary constraints on this glycolytic enzyme in these lineages.     

Different rates of substitution may produce different phylogenies of the eutherian mammals

Summary In an attempt to resolve some points of branching order in the phylogeny of the eutherian mammals, a phylogenetic analysis of 26 nuclear and 6 mitochondrial genes was undertaken using a maximum likelihood method on a constant rate stochastic model of molecular evolution. Seventeen of the nuclear genes gave a primates/artiodactyls grouping highest support whereas three of the mitochondrial genes found a rodents/artiodactyls grouping to be best supported. The primates/rodents grouping was never the best supported. On the assumption that rodents are indeed an outgroup to primates and artiodactyls and that the latter taxa diverged 70 million years ago, an estimation was made, for each gene, of the time of divergence of the rodent lineage. In most cases such estimates were beyond the limits set by present interpretations of the paleontological record as were many estimates of the divergence time of mouse and rat. These results suggest that, although there is locus variation, the divergent position of the rodent lineage may be an artifact of an elevated rate of nucleotide substitution in this order.     

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Summary In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.Abbreviations b endoplasmic reticulum extension, blebbing - er endoplasmic reticulum - f fibrillar material - g Golgi-like structure - h haustorium - hb haustorial body - hcw haustorial cell wall - hcy haustorial cytoplasm - hf haustorial finger - hocw host cell wall - hocy host cytoplasm - 1 lipid-like droplet - m mitochondrion - mt microtubule - mve multivesicular body - n nucleus - p papilla - ph penetration site of an infection peg - pl plasma membrane - s sheath - sm extrahaustorial membrane - v vacuole - ve vesicle