Role of an active site adenine in hairpin ribozyme catalysis

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.     

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.     

Twin ribozyme mediated removal of nucleotides from an internal RNA site

Over the past two decades, the structure and mechanism of catalytic RNA have been extensively studied; now ribozymes are understood well enough to turn them into useful tools. After we have demonstrated the twin ribozyme mediated insertion of additional nucleotides into a predefined position of a suitable substrate RNA, we here show that a similar type of twin ribozyme is also capable of mediating the opposite reaction: the site-specific removal of nucleotides. In particular, we have designed a twin ribozyme that supports the deletion of four uridine residues from a given RNA substrate. This reaction is a kind of RNA recombination that in the specific context of gene therapy mimics, at the level of RNA, the correction of insertion mutations. As a result of the twin ribozyme driven reaction, 17% of substrate are converted into the four nucleotides shorter product RNA.     

The beta subunit loop that couples catalysis and rotation in ATP synthase has a critical length

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix structure in the C-terminal domain of the β subunit containing the conserved DELSEED motif, termed “DELSEED-loop,” was suggested to be involved in coupling between catalysis and rotation. If this is indeed the role of the loop, it must have a critical length, the minimum length required to sustain its function. Here, the critical length of the DELSEED-loop was determined by functional analysis of mutants of Bacillus PS3 ATP synthase that had 7–14 amino acids within the loop deleted. A 10 residue deletion lost the ability to catalyze ATP synthesis, but was still an active ATPase. Deletion of 14 residues abolished any enzymatic activity. Modeling indicated that in both deletion mutants the DELSEED-loop was shortened by ∼10 Å; fluorescence resonance energy transfer experiments confirmed the modeling results. This appears to define the minimum length for DELSEED-loop required for coupling of catalysis and rotation. In addition, we could demonstrate that the loss of high-affinity binding to the catalytic site(s) that had been observed previously in two deletion mutants with 3–4 residues removed was not due to the loss of negative charged residues of the DELSEED motif in these mutants. An AALSAAA mutant in which all negative charges of the DELSEED motif were removed showed a normal pattern for MgATP binding to the catalytic sites, with a clearly present high-affinity site.     

Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization

Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.     

DNA lesion alters global conformational dynamics of Y-family DNA polymerase during catalysis

A major product of oxidative damage to DNA, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), can lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in vivo. However, our pre-steady-state kinetic studies show that DNA polymerase IV (Dpo4), a prototype Y-family enzyme from Sulfolobus solfataricus, can bypass 8-oxoG both efficiently and faithfully. For the first time, our stopped-flow FRET studies revealed that a DNA polymerase altered its synchronized global conformational dynamics in response to a DNA lesion. Relative to nucleotide incorporation into undamaged DNA, three of the four domains of Dpo4 undertook different conformational transitions during 8-oxoG bypass and the subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA induced by nucleotide binding was significantly hindered by the interactions between the embedded 8-oxoG and Dpo4 during the extension step. These results unprecedentedly demonstrate that a Y-family DNA polymerase employs different global conformational dynamics when replicating undamaged and damaged DNA.     

The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ～20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.     

Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P₋₁. Although RNase A also contains a P₋₁ subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P₋₁ subsite residues Arg³⁶, Asn³⁹, and Gln⁴⁰ of EDN in its catalytic activity. The individual mutation of Arg³⁶, Asn ³⁹, and Gln⁴⁰ resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P₋₁ subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates. Deepa Sikriwal and Divya Seth contributed equally to this work.     

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis

Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp¹²⁰ and Lys⁷⁷ is suggested.     

The solution structure of the VS ribozyme active site loop reveals a dynamic "hot-spot"

The VS ribozyme is the largest ribozyme in its class and is also the least structurally characterized thus far. The current working model of the VS ribozyme locates the active site in stem-loop VI. The solution structure of this active site loop was determined using high resolution NMR spectroscopy. The structure reveals that the ground-state conformation of the active site differs significantly from that determined previously from chemical structure probing and mutational analysis of the ribozyme in its active conformation, which contains several looped out bases. In contrast, the base-pairing scheme found for the isolated loop contains three mismatched base-pairs: an A+-C, a G-U wobble, and a sheared G-A base-pair and no looped out bases. Dynamics observed within the active site loop provide insight into the mechanism by which the RNA can rearrange its secondary structure into an "activated" conformation prior to cleavage. These findings lend support to the idea that RNA secondary structure is more fluid than once believed and that a better understanding of structure and dynamic features of ribozymes is required to unravel the intricacies of their catalytic abilities.     

Interaction of peptide substrate outside the active site influences catalysis by CaMKII

The interaction of calcium/calmodulin-dependent protein kinase II (CaMKII) with the NR2B subunit of N-methyl-D-aspartate-type glutamate receptor is thought to be one of the important events leading to synaptic plasticity. CaMKII binds NR2B by its catalytic site and by the autophosphorylation site binding pocket (APBP), a non-catalytic site. Mutagenesis of Glu-236, a residue in the APBP of CaMKII that is likely to be interacting with NR2B, influences phosphorylation of NR2B. The phosphorylation of syntide-2, a classical catalytic site substrate of CaMKII, is influenced to a much lesser extent by this mutation. Taken together these results indicate that interaction of NR2B at the non-catalytic site of CaMKII influences catalysis. Our data suggest that kinetic models of peptide substrate phosphorylation by CaMKII should incorporate the non-catalytic mode of binding of peptides that is dependent on the sequence of the peptide.     

Functional group requirements in the probable active site of the VS ribozyme

The VS ribozyme catalyses the site-specific cleavage of a phosphodiester linkage by a transesterification reaction that entails the attack of the neighbouring 2'-oxygen with departure of the 5'-oxygen. We have previously suggested that the A730 loop is an important component of the active site of the ribozyme, and that A756 is especially important in the cleavage reaction. Functional group modification experiments reported here indicate that the base of A756 is more important than its ribose for catalysis. A number of changes to the base, including complete ablation, lead to cleavage rates that are reduced 1000-fold, while removal of the 2'-hydroxyl group from the ribose results in tenfold slower cleavage. 2-Aminopurine fluorescence experiments indicate that this 2'-hydroxyl group is important for the structure of the A730 loop. Catalytic activity is especially sensitive to changes involving the exocyclic amine of A756; by contrast, the cleavage activity is only weakly sensitive to modification at the 7-position of the purine nucleus. These results suggest that the Watson-Crick edge of the adenine base is important in ribozyme function. We sought to test the possibility of a direct role of the nucleobase in the chemistry of the cleavage reaction. Addition of imidazole base in the medium failed to restore the activity of a ribozyme from which the nucleobase of A756 was removed. However, no restoration was obtained with exogenous adenine base either, indicating that the cavity that might result from ablation of the base was closed.     

Structural effects of nucleobase variations at key active site residue Ade38 in the hairpin ribozyme

The hairpin ribozyme requires functional groups from Ade38 to achieve efficient bond cleavage or ligation. To identify molecular features that contribute to catalysis, structures of position 38 base variants 2,6-diaminopurine (DAP), 2-aminopurine (AP), cytosine (Cyt), and guanine (Gua) were determined between 2.2 and 2.8 A resolution. For each variant, two substrate modifications were compared: (1) a 2'-O-methyl-substituent at Ade-1 was used in lieu of the nucleophile to mimic the precatalytic state, and (2) a 3'-deoxy-2',5'-phosphodiester linkage between Ade-1 and Gua+1 was used to mimic a reaction-intermediate conformation. While the global fold of each variant remained intact, the results revealed the importance of Ade38 N1 and N6 groups. Absence of N6 resulting from AP38 coincided with failure to localize the precatalytic scissile phosphate. Cyt38 severely impaired catalysis in a prior study, and its structures here indicated an anti base conformation that sequesters the imino moiety from the scissile bond. Gua38 was shown to be even more deleterious to activity. Although the precatalytic structure was nominally affected, the reaction-intermediate conformation indicated a severe electrostatic clash between the Gua38 keto oxygen and the pro-Rp oxygen of the scissile bond. Overall, position 38 modifications solved in the presence of 2'-OMe Ade-1 deviated from in-line geometry, whereas variants with a 2',5' linkage exhibited S-turn destabilization, as well as base conformational changes from syn to anti. These findings demonstrate the importance of the Ade38 Watson-Crick face in attaining a reaction-intermediate state and the sensitivity of the RNA fold to restructuring when electrostatic and shape features fail to complement.     

Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase

Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter sphaeroides (RsTAL). Kinetic analysis of RsTAL shows that the enzyme displays a 90-fold preference for L-tyrosine versus L-phenylalanine as a substrate. The pH-dependence of TAL activity with L-tyrosine and L-phenylalanine demonstrates a common protonation state for catalysis, but indicates a difference in charge-state for binding of either amino acid. Site-directed mutagenesis demonstrates that Ser150, Tyr60, and Tyr300 are essential for catalysis. Mutation of Ser150 to an alanine abrogates formation of the MIO prosthetic group, as shown by mass spectrometry, and prevents catalysis. The Y60F and Y300F mutants were inactive with both amino acid substrates, but bound p-coumaric and cinnamic acids with less than 12-fold changes in affinity compared the wild-type enzyme. Analysis of MIO-dithiothreitol adduct formation shows that the reactivity of the prosthetic group is not significantly altered by mutation of either Tyr60 or Tyr300. The mechanistic roles of Ser150, Tyr60, and Tyr300 are discussed in relation to the three-dimensional structure of RsTAL and related MIO-containing enzymes.     

Crystal structures of Staphylococcus epidermidis mevalonate diphosphate decarboxylase bound to inhibitory analogs reveal new insight into substrate binding and catalysis

The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 Å resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). Comparison of these structures provides a physical basis for the significant differences in K_i values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser¹⁹² as making potential contributions to catalysis. Significantly, Ser → Ala substitution of this side chain decreases k_cat by ∼10³-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 Å cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.     

Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe³⁺←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe³⁺-nitrile intermediate species are similar to low spin Fe³⁺-enzyme and model complexes bound by NO or N₃⁻. These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe³⁺ center.     

The origin of the RNA world: co-evolution of genes and metabolism

Discoveries demonstrating that RNA can serve genetic, catalytic, structural, and regulatory roles have provided strong support for the existence of an RNA World that preceded the origin of life as we know it. Despite the appeal of this idea, it has been difficult to explain how macromolecular RNAs emerged from small molecules available on the early Earth. We propose here a mechanism by which mutual catalysis in a pre-biotic network initiated a progression of stages characterized by ever larger and more effective catalysts supporting a proto-metabolic network, and the emergence of RNA as the dominant macromolecule due to its ability to both catalyze chemical reactions and to be copied in a template-directed manner. This model suggests that many features of modern life, including the biosynthetic pathways leading to simple metabolites, the structures of organic and metal ion cofactors, homochirality, and template-directed replication of nucleic acids, arose long before the RNA World and were retained as pre-biotic systems became more sophisticated.     

Association of novel domain in active site of archaic hyperthermophilic maltogenic amylase from Staphylothermus marinus

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.     

Ensemble perspective for catalytic promiscuity: calorimetric analysis of the active site conformational landscape of a detoxification enzyme

Enzymological paradigms have shifted recently to acknowledge the biological importance of catalytic promiscuity. However, catalytic promiscuity is a poorly understood property, and no thermodynamic treatment has described the conformational landscape of promiscuous versus substrate-specific enzymes. Here, two structurally similar glutathione transferase (GST, glutathione S-transferase) isoforms with high specificity or high promiscuity are compared. Differential scanning calorimetry (DSC) indicates a reversible low temperature transition for the promiscuous GSTA1-1 that is not observed with substrate-specific GSTA4-4. This transition is assigned to rearrangement of the C terminus at the active site of GSTA1-1 based on the effects of ligands and mutations. Near-UV and far-UV circular dichroism indicate that this transition is due to repacking of tertiary contacts with the remainder of the subunit, rather than "unfolding" of the C terminus per se. Analysis of the DSC data using a modified Landau theory indicates that the local conformational landscape of the active site of GSTA1-1 is smooth, with barrierless transitions between states. The partition function of the C-terminal states is a broad unimodal distribution at all temperatures within this DSC transition. In contrast, the remainder of the GSTA1-1 subunit and the GSTA4-4 protein exhibit folded and unfolded macrostates with a significant energy barrier separating them. Their partition function includes a sharp unimodal distribution of states only at temperatures that yield either folded or unfolded macrostates. At intermediate temperatures the partition function includes a bimodal distribution. The barrierless rearrangement of the GSTA1-1 active site within a local smooth energy landscape suggests a thermodynamic basis for catalytic promiscuity.     

A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.