One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems.

We have determined the pKA values of the 12 carboxyl residues in the native and denatured state of barnase by a combination of thermodynamic measurements on mutants of charged residues and NMR titration data. The pKA values of the 11 residues titrating under folding conditions (above pH 2.2) were determined by two-dimensional 1H NMR. The pKA value of the remaining residue, Asp 93 which forms a salt link with Arg 69 and titrates at much lower pH values, was determined by changes in the pH dependence of the stability of the protein upon mutation to Asn: pKAsp93A at low ionic strength (50 mM) and pKAsp93A at high ionic strength (600 mM). The overall titration of the native state is nonideal, and the protein retains fractionally ionized residues other than Asp 93 throughout the experimental pH range of 0.2-6.3. Protonation events taking place at pH values below 2 were further characterized by the pH dependence of the unfolding kinetics of wild-type and charge-mutant proteins. By comparing the observed pH dependence of the protein stability with that calculated from the pKA values for the native protein, we demonstrate that the pKA values of the denatured state are significantly lower than those reported for model compounds: the pKA values of the denatured state appear on average 0.4 units lower than previous estimates in the presence of chemical denaturant. The results have direct implications for calculations of the energetics of proton equilibria and suggest that the acid/thermally denatured state is not an extended coil where the residues are isolated from one another by the intervening solvent but is compact and involves intramolecular charge repulsion.

The increase in enzymatic rates with temperature up to an optimum temperature (Topt) is widely attributed to classical Arrhenius behavior, with the decrease in enzymatic rates above Topt ascribed to protein denaturation and/or aggregation. This account persists despite many investigators noting that denaturation is insufficient to explain the decline in enzymatic rates above Topt. Here we show that it is the change in heat capacity associated with enzyme catalysis (ΔC(‡)p) and its effect on the temperature dependence of ΔG(‡) that determines the temperature dependence of enzyme activity. Through mutagenesis, we demonstrate that the Topt of an enzyme is correlated with ΔC(‡)p and that changes to ΔC(‡)p are sufficient to change Topt without affecting the catalytic rate. Furthermore, using X-ray crystallography and molecular dynamics simulations we reveal the molecular details underpinning these changes in ΔC(‡)p. The influence of ΔC(‡)p on enzymatic rates has implications for the temperature dependence of biological rates from enzymes to ecosystems.

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Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory -MMRT (Hobbs et al., 2013, ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q 10 calculations) at low temperatures and that Q 10 declined as temperature increases in agreement with data synthesis from the literature. Declining Q 10 and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme-catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems -a fundamental component of models for climate change.

Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 Å resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg 2؉ -dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNA i and nonsense-mediated RNA degradation.The explosive growth of whole genome sequencing efforts, and the discovery that a large proportion of the assumed gene products are of unknown or poorly understood function, has focused attention on new approaches to assigning function. In the absence of sufficient sequence similarity to clearly infer homology with already characterized proteins, a variety of bioinformatic approaches have been used to obtain functional clues. These include, for example, analyses of genome location (seeking potential operons), phylogenetic profiling, and observations of gene fusions in different species (1, 2). An alternative, complementary, approach is to use analyses of protein three-dimensional structure to derive functional insights, because three-dimensional structure is conserved in evolution much more strongly than sequence. This provides a rationale for a number of structural genomics initiatives (3-6).As part of a pilot structural genomics project aimed at the discovery of biological function, we have focused on gene products from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, an organism whose complete genome sequence was published recently (7). A whole-genome comparison of P. aerophilum and Mycobacterium tuberculosis, two organisms with very different, and in a sense extreme, lifestyles, led us to identify a set of 250 pairs of orthologous genes that are both widely distributed in nature and are shared by these two organisms. Among these were a set of four genes from P. aerophilum (PAE0151, PAE0285, PAE0337, and PAE2754) and four from M. tuberculosis (Rv0065, Rv0549, Rv0960, and Rv1720) that have since be...

The PIN-domains are small proteins of ~130 amino acids that are found in bacteria, archaea and eukaryotes and are defined by a group of three strictly conserved acidic amino acids. The conserved three-dimensional structures of the PIN-domains cluster these acidic residues in an enzymatic active site. PIN-domains cleave single-stranded RNA in a sequence-specific, Mg²+- or Mn²+-dependent manner. These ribonucleases are toxic to the cells which express them and to offset this toxicity, they are co-expressed with tight binding protein inhibitors. The genes encoding these two proteins are adjacent in the genome of all prokaryotic organisms where they are found. This sequential arrangement of inhibitor-RNAse genes conforms to that of the so-called toxin-antitoxin (TA) modules and the PIN-domain TAs have been named VapBC TAs (virulence associated proteins, VapB is the inhibitor which contains a transcription factor domain and VapC is the PIN-domain ribonuclease). The presence of large numbers of vapBC loci in disparate prokaryotes has motivated many researchers to investigate their biochemical and biological functions. For example, the devastating human pathogen Mycobacterium tuberculosis has 45 vapBC loci encoded in its genome whereas its non-pathogenic relative, Mycobacterium smegmatis has just one vapBC operon. On another branch of the prokaryotic tree, the nitrogen-fixing symbiont of legumes, Sinorhizobium meliloti has 21 vapBC loci and at least one of these loci have been implicated in the regulation of growth in the plant nodule. A range of biological functions has been suggested for these operons and this review sets out to survey the PIN-domains and summarise the current knowledge about the vapBC TA systems and their roles in diverse bacteria.

Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed, along with changes in energetic fluctuations across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.