Role In Transition State Stabilization Research Articles

Structural Characterization of Arginine Fingers: Identification of an Arginine Finger for the Pyrophosphatase dUTPases.

Arginine finger is a highly conserved and essential residue in many GTPase and AAA+ ATPase enzymes that completes the active site from a distinct protomer, forming contacts with the γ-phosphate of the nucleotide. To date, no pyrophosphatase has been identified that employs an arginine finger fulfilling all of the above properties; all essential arginine fingers are used to catalyze the cleavage of the γ-phosphate. Here, we identify and unveil the role of a conserved arginine residue in trimeric dUTPases that meets all the criteria established for arginine fingers. We found that the conserved arginine adjacent to the P-loop-like motif enables structural organization of the active site for efficient catalysis via its nucleotide coordination, while its direct electrostatic role in transition state stabilization is secondary. An exhaustive structure-based comparison of analogous, conserved arginines from nucleotide hydrolases and transferases revealed a consensus amino acid location and orientation for contacting the γ-phosphate of the substrate nucleotide. Despite the structurally equivalent position, functional differences between arginine fingers of dUTPases and NTPases are explained on the basis of the unique chemistry performed by the pyrophosphatase dUTPases.

Ground-State Destabilization by Phe-448 and Phe-449 Contributes to Tyrosine Phenol-Lyase Catalysis

The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme–substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant tyrosine phenol-lyase (TPL) form complexes with 3-fluoro-l-tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20° out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigate...

Structure and mechanism of E. coli RNA 2',3'-cyclic phosphodiesterase.

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD, which was identified originally as a reversible transesterifying “nuclease/ligase” at RNA 2′,5′-phosphodiesters. We find that YapD is an “end healing” cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an HORNA>p substrate with a 2′,3′-cyclic phosphodiester to a HORNAp product with a 2′-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2′,3′-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2′-AMP highlights the roles of extended histidine-containing motifs 43HxTxxF48 and 125HxTxxR130 in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2′-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2′-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π–π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.

Spectroscopic and Electronic Structure Studies Probing Covalency Contributions to C–H Bond Activation and Transition-State Stabilization in Xanthine Oxidase

A detailed electron paramagnetic resonance (EPR) and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed and serves as a basis for developing a valence-bond description of C-H activation and transition-state (TS) stabilization along the reaction coordinate with aldehyde substrates. EPR spectra of aldehyde-inhibited XO have been analyzed in order to provide information regarding the relationship between the g, (95,97)Mo hyperfine (A(Mo)), and (13)C hyperfine (A(C)) tensors. Analysis of the EPR spectra has allowed for greater insight into the electronic origin of key delocalizations within the Mo-O(eq)-C fragment and how these contribute to C-H bond activation/cleavage and TS stabilization. A natural bond orbital analysis of the enzyme reaction coordinate with aldehyde substrates shows that both Mo═S π → C-H σ* (ΔE = 24.3 kcal mol(-1)) and C-H σ → Mo═S π* (ΔE = 20.0 kcal mol(-1)) back-donation are important in activating the substrate C-H bond for cleavage. Additional contributions to C-H activation derive from O(eq) lp → C-H σ* (lp = lone pair; ΔE = 8.2 kcal mol(-1)) and S lp → C-H σ* (ΔE = 13.2 kcal mol(-1)) stabilizing interactions. The O(eq)-donor ligand that derives from water is part of the Mo-O(eq)-C fragment probed in the EPR spectra of inhibited XO, and the observation of O(eq) lp → C-H σ* back-donation indicates a key role for O(eq) in activating the substrate C-H bond for cleavage. We also show that the O(eq) donor plays an even more important role in TS stabilization. We find that O(eq) → Mo + C charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal mol(-1)) and the Mo-O(eq)-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-O(eq)-C delocalization at the TS allows for the TS to be described in valence-bond terms as a resonance hybrid of the reactant (R) and product (P) valence-bond wave functions.

Design of Biomimetic Catalysts by Molecular Imprinting in Synthetic Polymers: The Role of Transition State Stabilization

The impressive efficiency and selectivity of biological catalysts has engendered a long-standing effort to understand the details of enzyme action. It is widely accepted that enzymes accelerate reactions through their steric and electronic complementarity to the reactants in the rate-determining transition states. Thus, tight binding to the transition state of a reactant (rather than to the corresponding substrate) lowers the activation energy of the reaction, providing strong catalytic activity. Debates concerning the fundamentals of enzyme catalysis continue, however, and non-natural enzyme mimics offer important additional insight in this area. Molecular structures that mimic enzymes through the design of a predetermined binding site that stabilizes the transition state of a desired reaction are invaluable in this regard. Catalytic antibodies, which can be quite active when raised against stable transition state analogues of the corresponding reaction, represent particularly successful examples. Recently, synthetic chemistry has begun to match nature's ability to produce antibody-like binding sites with high affinities for the transition state. Thus, synthetic, molecularly imprinted polymers have been engineered to provide enzyme-like specificity and activity, and they now represent a powerful tool for creating highly efficient catalysts. In this Account, we review recent efforts to develop enzyme models through the concept of transition state stabilization. In particular, models for carboxypeptidase A were prepared through the molecular imprinting of synthetic polymers. On the basis of successful experiments with phosphonic esters as templates to arrange amidinium groups in the active site, the method was further improved by combining the concept of transition state stabilization with the introduction of special catalytic moieties, such as metal ions in a defined orientation in the active site. In this way, the imprinted polymers were able to provide both an electrostatic stabilization for the transition state through the amidinium group as well as a synergism of transition state recognition and metal ion catalysis. The result was an excellent catalyst for carbonate hydrolysis. These enzyme mimics represent the most active catalysts ever prepared through the molecular imprinting strategy. Their catalytic activity, catalytic efficiency, and catalytic proficiency clearly surpass those of the corresponding catalytic antibodies. The active structures in natural enzymes evolve within soluble proteins, typically by the refining of the folding of one polypeptide chain. To incorporate these characteristics into synthetic polymers, we used the concept of transition state stabilization to develop soluble, nanosized carboxypeptidase A models using a new polymerization method we term the "post-dilution polymerization method". With this methodology, we were able to prepare soluble, highly cross-linked, single-molecule nanoparticles. These particles have controlled molecular weights (39 kDa, for example) and, on average, one catalytically active site per particle. Our strategies have made it possible to obtain efficient new enzyme models and further advance the structural and functional analogy with natural enzymes. Moreover, this bioinspired design based on molecular imprinting in synthetic polymers offers further support for the concept of transition state stabilization in catalysis.

A Combined Molecular Dynamics and Rapid Kinetics Approach to Identify Conserved Three-Dimensional Communication Networks in Elongation Factor Tu

Elongation factor (EF) Tu delivers aminoacyl-tRNAs to the actively translating bacterial ribosome in a GTP-hydrolysis-dependent process. Rapid recycling of EF-Tu, catalyzed by EF-Ts, is required for efficient protein synthesis in vivo. Here we report a combined theoretical and experimental approach aimed at identifying three-dimensional communication networks in EF-Tu. As an example, we focus on the mechanistic role of second-shell residue Asp109. We constructed full-length structural models of EF-Tu from Escherichia coli in the GDP-/GTP-bound state and performed several 10-ns-long molecular-dynamics simulations. During these simulations, the side chain of Asp109 formed a previously undetected transient hydrogen bond to His22, an invariant residue in the phosphate-binding loop (P-loop). To experimentally validate our molecular-dynamics results and further analyze the role of this hydrogen bond, we determined all rate constants for the multistep reaction between EF-Tu (wild-type and two mutants), EF-Ts, GDP, and GTP using the stopped-flow technique. This mutational analysis revealed that the side chain of Asp109 is important for acceleration of GDP, but not for GTP dissociation by EF-Ts. The possibility that the Asp109 side chain has a role in transition-state stabilization and coupling of P-loop movements with rearrangements at the base side of the nucleotide is discussed.

Mechanistic Insight into the Role of Transition-State Stabilization in Cyclophilin A

Peptidyl prolyl cis−trans isomerases (PPIases) are ubiquitous enzymes in biology that catalyze the cis−trans isomerization of the proline imide peptide bond in many cell signaling pathways. The local change of the isomeric state of the prolyl peptide bond acts as a switching mechanism in altering the conformation of proteins. A complete understanding of the mechanism of PPIases is still lacking, and current experimental techniques have not been able to provide a detailed atomistic picture. Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis−trans isomerization of the free and cyclophilin A-catalyzed process. We show that the catalytic mechanism of cyclophilin is due mainly to the stabilization and preferential binding of the transition state that is achieved by a favorable hydrogen bond interaction with a backbone NH group. We also show that the substrate in the transition state interacts more favorably with the enzyme than the cis isomer, which in turn interacts more favorably than the trans isomer. The stability of the enzyme−substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue. Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

Hydrogen bond residue positioning in the 599–611 loop of thimet oligopeptidase is required for substrate selection

Thimet oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of thimet oligopeptidase in which Gly or Tyr residues in the 599-611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in k(cat) observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.

Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase.

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.

456 Caelyx (pegylated liposomal doxorubicin hcl) and conventional doxorubicin have significantly different adverse event profiles

Despite its small size, the 205 nt group I intron from Azoarcus tRNAIle is an exceptionally stable self-splicing RNA. This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices. These features make it an ideal system to establish the conserved chemical basis of group I intron activity. We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups. In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a “chemical phylogeny” of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts. The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor. These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons. The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5′-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2′-OH groups for P1 helix docking into most of the single stranded segment J8/7. However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5′-end of the J8/7. Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction. This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization.

Catalytic Acid−Base Groups in Yeast Pyruvate Decarboxylase. 1. Site-Directed Mutagenesis and Steady-State Kinetic Studies on the Enzyme with the D28A, H114F, H115F, and E477Q Substitutions

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions. Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Mechanism of hydrolysis of phosphate esters by the dimetal center of 5′-nucleotidase based on crystal structures

5′-Nucleotidase belongs to a large superfamily of distantly related dinuclear metallophosphatases including the Ser/Thr protein phosphatases and purple acid phosphatases. The protein undergoes a 96° domain rotation between an open (inactive) and a closed (active) enzyme form. Complex structures of the closed form with the products adenosine and phosphate, and with the substrate analogue inhibitor α,β-methylene ADP, have been determined at 2.1 Å and 1.85 Å resolution, respectively. In addition, a complex of the open form of 5′-nucleotidase with ATP was analyzed at a resolution of 1.7 Å. These structures show that the adenosine group binds to a specific binding pocket of the C-terminal domain. The adenine ring is stacked between Phe429 and Phe498. The N-terminal domain provides the ligands to the dimetal cluster and the conserved His117, which together form the catalytic core structure. However, the three C-terminal arginine residues 375, 379 and 410, which are involved in substrate binding, may also play a role in transition-state stabilization. The β-phosphate group of the inhibitor is terminally coordinated to the site 2 metal ion. The site 1 metal ion coordinates a water molecule which is in an ideal position for a nucleophilic attack on the phosphorus atom, assuming an in-line mechanism of phosphoryl transfer. Another water molecule bridges the two metal ions.

Kinetic characterization of the second step of group II intron splicing: role of metal ions and the cleavage site 2'-OH in catalysis.

The ai5gamma group II intron from yeast excises itself from precursor transcripts in the absence of proteins. When a shortened form of the intron containing all but the 3'-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3' splice site oligonucleotide, a nucleotidyl transfer reaction occurs that mimics the second step of splicing. As this tripartite reaction provides a means to identify important functional groups in 3' splice site recognition and catalysis, we establish here a minimal kinetic framework and demonstrate that the chemical step is rate-limiting. We use this framework to characterize the metal ion specificity switch observed previously upon sulfur substitution of the 3'-oxygen leaving group and to elucidate by atomic mutagenesis the role of the neighboring 2'-OH in catalysis. The results suggest that both the 3'-oxygen leaving group and the neighboring 2'-OH are important ligands for metal ions in the transition state but not in the ground state and that the 2'-OH may play an additional role in transition state stabilization by donating a hydrogen bond. Metal specificity switch experiments combined with quantitative analysis show that the Mn(2+) that interacts with the leaving group binds to the ribozyme with the same affinity as the metal ion that interacts with the neighboring 2'-OH, raising the possibility that a single metal ion mediates interactions with the 2'- and 3'-oxygen atoms at the 3' splice site.

A chemical phylogeny of group I introns based upon interference mapping of a bacterial ribozyme

Despite its small size, the 205 nt group I intron from Azoarcus tRNAIle is an exceptionally stable self-splicing RNA. This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices. These features make it an ideal system to establish the conserved chemical basis of group I intron activity. We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups. In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a “chemical phylogeny” of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts. The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor. These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons. The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5′-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2′-OH groups for P1 helix docking into most of the single stranded segment J8/7. However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5′-end of the J8/7. Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction. This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization.

Role of Arginine 277 in (S)-Mandelate Dehydrogenase from Pseudomonas putida in Substrate Binding and Transition State Stabilization

(S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase. Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group [Lindqvist, Y., Branden, C.-I., Mathews, F. S., and Lederer, F. (1991) J. Biol. Chem. 266, 3198-3207]. The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments. The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K. The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value. Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents. Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate. The positive charge has a relatively small contribution toward substrate binding. R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate.

The strongly conserved lysine 256 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase is essential for phosphoryl transfer.

Lysine 256, a conserved amino acid of Saccharomycescerevisiae phosphoenolpyruvate (PEP) carboxykinase located in the consensus kinase 1a sequence of the enzyme, was changed to alanine, arginine, or glutamine by site-directed mutagenesis. These substitutions did not result in gross changes in the protein structure, as indicated by circular dichroism, tryptophan fluorescence spectroscopy, and gel-exclusion chromatography. The three variant enzymes showed almost unaltered Km for MnADP but about a 20 000-fold decrease in Vmax for the PEP carboxylation reaction, as compared to wild-type PEP carboxykinase. The variant enzymes presented oxaloacetate decarboxylase activity at levels similar to those of the native protein; however, they lacked pyruvate kinase-like activity. The dissociation constant for the enzyme-MnATP complex was 1.3 +/- 0.3 microM for wild-type S. cerevisiae PEP carboxykinase, and the corresponding values for the Lys256Arg, Lys256Gln, and Lys256Ala mutants were 2.0 +/- 0.6 microM, 17 +/- 2 microM, and 20 +/- 6 microM, respectively. These results collectively show that a positively charged residue is required for proper binding of MnATP and that Lys256 plays an essential role in transition state stabilization during phosphoryl transfer for S. cerevisiae PEP carboxykinase.

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2′-hydroxyl groups

Background: Group II introns are self-splicing RNAs that have mechanistic similarity to the spliceosome complex involved in messenger RNA splicing in eukaryotes. These autocatalytic molecules can be recon-figured into highly specific, multiple-turnover ribozyines that cleave oligonucleorides in trans. We set out to use a simplified system of this kind to study the mechanisin of cleavage. Results: Unlike other catalytic RNA molecules, the group II ribozymes cleave DNA linkages almost as readily as RNA linkages. One ribozyme variant cleaves DNA linkages with an efficiency comparable to that of restriction endonuclease EcoRI. Single deoxynucleotide substitutions in the substrate showed that the ribozymes bind substrate without engaging 2′-hydroxyl groups. Conclusions: The ribose 2′-hydroxyl group at the cleavage site has little role in transition-state stabilization by group II ribozymes. Substrate 2′-hydroxyl groups are not involved in substrate binding, suggesting that only base-pairing is required for substrate recognition.

Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization.

The pH dependence of cathepsin B-catalyzed hydrolyzes is very complex. At least seven dissociable groups are involved in the binding and hydrolysis of 7-amido-4-methyl coumarin and p-nitroaniline (pNA)-based substrates containing a P1 Arg and either a Phe or Arg at the P2 position. By site-directed mutagenesis we show that a previous suggestion, that Arg202 is one of the groups which influences the pH dependence of cathepsin B-catalyzed hydrolysis of the Z-Arg-Arg-pNA substrate, is not valid. However, it was found that Glu245, which has a pKa of 5.1 in rat cathepsin B, is responsible for the S2-P2 specificity for Arg-containing substrates and controls the pH dependence of their hydrolysis. Furthermore, the data indicate that Glu245, which forms a hydrogen bond with the guanidinium group of the substrate's P2 Arg, contributes about 1.8 kcal/mol to transition state stabilization in the protonated state and about 0.6 kcal/mol in the deprotonated state. Mutation of Glu245 to Gln results in a 16-fold decrease in kcat but does not affect Km. While cathepsin B has a 7-fold preference for Phe over Arg at the P2 position of a substrate, binding of the aromatic side chain does not appear to be influenced by Glu245.

Refined crystal structure of the phosphorylase-heptulose 2-phosphate-oligosaccharide-AMP complex

The crystal structure of phosphorylase b-heptulose 2-phosphate complex with oligosaccharide and AMP bound has been refined by molecular dynamics and crystallographic least-squares with the program XPLOR. Shifts in atomic positions of up to 4 Å from the native enzyme structure were correctly determined by the program without manual intervention. The final crystallographic R value for data between 8 and 2.86 Å resolution is 0.201, and the overall root-mean-square difference between the native and complexed structure is 0.58 Å for all protein atoms. The results confirm the previous observation that there is a direct hydrogen bond between the phosphate of heptulose 2-phosphate and the pyridoxal phosphate 5′-phosphate group. The close proximity of the two phosphates is stabilized by an arginine residue, Arg569, which shifts from a site buried in the protein to a position where it can make contact with the product phosphate. There is a mutual interchange in position between the arginine and an acidic group, Asp283. These movements represent the first stage of the allosteric response which converts the catalytic site from a low to a high-affinity binding site. Communication of these changes to other sites is prevented in the crystal by the lattice forces, which also form the subunit interface. The constellation of groups in the phosphorylase transition state analogue complex provides a structural basis for understanding the catalytic mechanism in which the cofactor pyridoxal phosphate 5′-phosphate group functions as a general acid to promote attack by the substrate phosphate on the glycosidic bond when the reaction proceeds in the direction of glycogen degradation. In the direction of glycogen synthesis, stereoelectronic effects contribute to the cleavage of the C-1O-1 bond. In both reactions the substrate phosphate plays a key role in transition state stabilization. The details of the oligosaccharide, maltoheptaose, interactions with the enzyme at the glycogen storage site are also described.