Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semi-empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hypersurfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.     

Ultrafast catalytic processes in enzymes

The study of biocatalysis and biotransformation in the transition-state region has been challenging and difficult, but recent advances on two important photoenzymes in nature, DNA photolyase and protochlorophyllide oxidoreductase, have enabled the investigation of their catalytic processes in real time. By following the entire evolution of substrate transformation, the functional dynamics constituting a series of elementary reactions have been mapped out. The five fundamental reactions in the enzymes, namely electron transfer, bond breaking and making, proton and hydride transfer, all occur ultrafast within subnanosecond. The direct clocking of catalytic transition states probes central, unmasked chemical processes and provides mechanistic insights into the role of the dynamics in enzyme function, which not only facilitates the formation of the enzyme-substrate complex in the transition-state configurations, but also modulates the subsequent catalytic reactions for maximum biotransformation efficiency.     

Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation

The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining step of the catalytic process is not the nucleophilic attack leading to the cycle formation (a very easy and fast process with a negligible barrier of 0.8 kcal mol(-1)), but a proton transfer, which is assisted by the Glu201 residue acting as a proton shuttle (general base and general acid). A complex network of hydrogen bonds (involving Asp248 and other residues) contribute to lower the activation barrier for the proton shift which affords the formation of an ammonia molecule bonded to the substrate. The ammonia molecule is a good leaving group which is easily expelled from the substrate in the last step of the catalytic cycle, but remains anchored to the enzyme as a ligand of the zinc cation. The metal plays a key-role in assisting the nucleophilic attack (electrostatic catalysis) since it polarizes the substrate gamma-amide carbonyl group (its electrophilic character increases). Also, the strength of the nucleophilic nitrogen (substrate alpha-amino group) is enhanced by hydrogen bonds involving the Glu201 residue. The computations outline the important role of Trp329 in helping the substrate binding process and stabilizing the cyclization transition state.     

The role of serine-246 in cytochrome P450eryF-catalyzed hydroxylation of 6-deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.     

The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation of 6-Deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronol ide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed participate in the proton shuttling pathway, and also support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction. Copyright 2000 Academic Press.     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

The catalytic cycle of catechol oxidase

Hybrid density functional theory with the B3LYP functional has been used to investigate the catalytic mechanism of catechol oxidase. Catechol oxidase belongs to a class of enzymes that has a copper dimer with histidine ligands at the active site. Another member of this class is tyrosinase, which has been studied by similar methods previously. An important advantage for the present study compared to the one for tyrosinase is that X-ray crystal structures exist for catechol oxidase. The most critical step in the mechanism for catechol oxidase is where the peroxide O–O bond is cleaved. In the suggested mechanism this cleavage occurs in concert with a proton transfer from the substrate. Shortly after the transition state is passed there is another proton transfer from the substrate, which completes the formation of a water molecule. An important feature of the mechanism, like the one for tyrosinase, is that no proton transfers to or from residues outside the metal complex are needed. The calculated energetics is in reasonable agreement with experiments. Comparisons are made to other similar enzymes studied previously.     

Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis.

The role of tyrosine 158 (Y158) and lysine 165 (K165) in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, has been investigated. These residues have been identified as putative catalytic residues on the basis of structural and sequence homology with the short chain alcohol dehydrogenase family of enzymes. Replacement of Y158 with phenylalanine (Y158F) and with alanine (Y158A) results in 24- and 1500-fold decreases in k(cat), respectively, while leaving K(m) for the substrate, trans-2-dodecenoyl-CoA, unaffected. Remarkably, however, replacement of Y158 with serine (Y158S) results in an enzyme with wild-type activity. Kinetic isotope effect studies indicate that the transfer of a solvent-exchangeable proton is partially rate-limiting for the wild-type and Y158S enzymes, but not for the Y158A enzyme. These data indicate that Y158 does not function formally as a proton donor in the reaction but likely functions as an electrophilic catalyst, stabilizing the transition state for hydride transfer by hydrogen bonding to the substrate carbonyl. A conformational change involving rotation of the Y158 side chain upon binding of the enoyl substrate to the enzyme is proposed as an explanation for the inverse solvent isotope effect observed on V/K(DD-CoA) when either NADH or NADD is used as the reductant. These data are consistent with the recently published structure of a C16 fatty acid substrate bound to InhA that shows Y158 hydrogen bonded to the substrate carbonyl group and rotated from the position it occupies in the InhA-NADH binary complex [Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589]. Finally, the role of K165 has been analyzed using site-directed mutagenesis. Replacement of K165 with glutamine (K165Q) and arginine (K165R) has no effect on the enzyme's catalytic ability or on its ability to bind NADH. However, the K165A and K165M enzymes are unable to bind NADH, indicating that K165 has a primary role in cofactor binding.     

Unusual four-bond secondary H/D isotope effect supports a short-strong hydrogen bond between phospholipase A2 and a transition state analogue inhibitor

A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA(2) in the presence of a phosphonate transition state analogue. The cross-modulation of H(epsilon2)/H48 and H(delta1)/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H(2)O/D(2)O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA(2), the observation that H(delta1)/H48 induces a 3-fold larger H/D secondary isotope effect ( approximately 0.15 ppm) on H(epsilon2)/H48 than vice versa ( approximately 0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short-strong hydrogen bond formed between the donor N(delta1)/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA(2) and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.     

Mechanism of peptide bond formation on the ribosome

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

Common Hydrogen Bond Interactions in Diverse Phosphoryl Transfer Active Sites

Phosphoryl transfer reactions figure prominently in energy metabolism, signaling, transport and motility. Prior detailed studies of selected systems have highlighted mechanistic features that distinguish different phosphoryl transfer enzymes. Here, a top-down approach is developed for comparing statistically the active site configurations between populations of diverse structures in the Protein Data Bank, and it reveals patterns of hydrogen bonding that transcend enzyme families. Through analysis of large samples of structures, insights are drawn at a level of detail exceeding the experimental precision of an individual structure. In phosphagen kinases, for example, hydrogen bonds with the O_3β of the nucleotide substrate are revealed as analogous to those in unrelated G proteins. In G proteins and other enzymes, interactions with O_3β have been understood in terms of electrostatic favoring of the transition state. Ground state quantum mechanical calculations on model compounds show that the active site interactions highlighted in our database analysis can affect substrate phosphate charge and bond length, in ways that are consistent with prior experimental observations, by modulating hyperconjugative orbital interactions that weaken the scissile bond. Testing experimentally the inference about the importance of O_3β interactions in phosphagen kinases, mutation of arginine kinase Arg₂₈₀ decreases k_cat, as predicted, with little impact upon K_M.     

Interactions between Asp, His, Ser residues within models of the active site of serine proteases. A theoretical empirical study

Empirical theoretical calculations have been performed on a simplified model of the active site of two serine proteases: alpha-chymotrypsin and subtilisin Novo. The stability of the catalytic triad and the hydrogen bond formation between the Asp-His and His-Ser pairs have been examined for different protonation states. The results show that the Asp-His interactions prevail upon the His-Ser ones. Agreement between calculated configurations and the crystal structure of the site suggests that the presence of other residues near the functional residues is not determinant for the stability of the triad in alpha-chymotrypsin. In subtilisin Novo, on the contrary, the presence of the neighbouring residues seems to contribute more largely to the stability. Strong hydrogen bond interactions between the His and Ser residues do not exist in the resting enzymes. Any improvement of the His-Ser interactions requires large destabilization of the Asp-His diad. Our results suggest that the mechanism of the proton transfer can occur only from perturbations of the active site structure induced by the presence of the substrate.     

Oxidation and electronic state dependence of proton transfer in the enzymatic cycle of cytochrome P450eryF

Hydrogen bond networks, consisting of hydrogen bonded waters anchored by polar/acidic amino acid sidechains, are often present in the vicinity of the oxygen binding clefts of P450s. Density functional and quantum dynamics calculations of a O(2) binding cleft network model of cytochrome P450eryF(CYP107A1) indicate that such structural motifs facilitate ultrafast proton transfer from network waters to the dioxygen of the reduced oxyferrous species via a multiple proton translocation mechanism with barriers of 7-10 kcal/mol on its doublet ground state, and that the energies of the proton transfer reactant and constrained proton transfer products have an electronic and oxidation state dependence [J. Am. Chem. Soc. 124 (2002) 1430]. In the present study, the origin of the oxidation state dependence is shown to have its roots in differential proton affinities while the electronic state dependence of the reduced oxyferrous heme has its origins in subtle differences in network topologies near the transition state of the initial proton transfer event. Relaxed potential surface scans and unconstrained proton transfer product optimizations indicate that the proton transfer product in both the singlet oxyferrous heme and the reduced oxyferrous heme species in a quartet state are not viable stable (bound) states relative to the reactant form. While the proton affinity of H(3)O(+) is sufficient for it to protonate both the oxyferrous and the reduced oxyferrous heme species, hydrogen bond network stabilized water is only capable of protonating the reduced oxyferrous form. This interpretation is substantiated by study of the NO bound reduced ferrous heme of P450nor, which is isoelectronic with the oxyferrous heme and has a similar proton affinity. Density functional calculations on a more extensive O(2) binding cleft model support the multiple proton translocation mechanism of transfer but indicates that the significant negative charge density on the bound dioxygen of the reduced oxyferrous heme species, in its doublet ground state, polarizes the associated hydrogen bond network sufficiently so as to result in short, strong, low-barrier hydrogen bonds. The computed O-H-O bond distances are less than 2.55 A and have a near degeneracy of the proton transfer reactant and initial (sudden) proton transfer products. These low-barrier hydrogen bond features, in addition to the finding of a (zero point uncorrected) barrier of 1.3 kcal/mol, indicate that proton transfer from water to the distal oxygen should be rapid, facile and may not require large curvature tunneling as originally suggested by use of a smaller model. An initial assessment of protonation of the reduced oxyferrous heme distal oxygen by a model of 6-deoxyerythronolide B (6-DEB) indicates it to be low barrier (3.8 kcal/mol) and exothermic (-2.9 kcal/mol). The combined results indicate the plausibility of simultaneous diprotonation of the distal oxygen of the reduced oxyferrous heme, leading to O-O bond scission, using the combined water network and 6-DEB substrate protonation agents.     

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex.

Molecular recognition and site-directed mutagenesis are used in combination to identify kinetically, transition state interactions between glucoamylase (GA) and the substrate maltose. Earlier studies of mutant Glu180----Gln GA had indicated a role in substrate binding for Glu180 (Sierks, M.R., Ford, C., Reilly, P.J. and Svensson, B. (1990) Protein Engng, 3, 193-198). Here, changes in activation energies calculated from measured kcat/Km values for a series of deoxygenated maltose analogues indicate hydrogen bonding between the mutant enzyme and the 3-OH group of the reducing end sugar ring. Using the same substrate analogues and determining activation energies with wild-type GA an additional hydrogen bond with the 2-OH group of maltose is attributed to an interaction with the carboxylate Glu180. This novel combination of molecular recognition and site-directed mutagenesis enables an enzyme substrate transition state contact to be identified and characterized even without access to the three dimensional structure of the enzyme. Given the distant structural relationships between glucoamylases and several starch hydrolases (Svensson,B. (1988) FEBS Lett., 230, 72-76), such identified contacts may ultimately guide tailoring of the activity of these related enzymes.     

The catalytic mechanism of Drosophila alcohol dehydrogenase: evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ ribose OH switch

The ionization properties of the active site residues in Drosophila lebanonensis alcohol dehydrogenase (DADH) were investigated theoretically by using an approach developed to account for multiple locations of the hydrogen atoms of the titratable and polar groups. The electrostatic calculations show that (a) the protonation/deprotonation transition of the binary complex of DADH is related to the coupled ionization of Tyr151 and Lys155 in the active site and (b) the pH dependence of the proton abstraction is correlated with a reorganization of the hydrogen bond network in the active site. On this basis, a proton relay mechanism for substrate dehydrogenation is proposed in which the O2' ribose hydroxyl group from the coenzyme has a key role and acts as a switch. The proton relay chain includes the active site catalytic residues, as well as a chain of eight water molecules that connects the active site with the bulk solvent.     

A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases.

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.     

Proton tunneling and enzyme catalysis

Summary It is proposed in this paper that enzymes, by virtue of a number of correctly positioned sites of interaction with substrates, can force the compression of hydrogen bonds, increasing the probability of proton transfer by quantum mechanical tunneling. By such a catalytic mechanism a rate enhancement of many orders of magnitude may be obtained with a very low energy input requirement. The mechanism would, however, require a highly structured catalyst.Pertinent aspects of hydrogen bond theory and of tunneling theory are briefly reviewed.Work supported by NIGMS Training Grant No. GM 678-07.     

Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Trans-sialidase is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. This enzyme catalyzes the transfer of sialic acids from mammalian host cells to parasitic cell surfaces in order to mask the infection from the host's immune system. It represents a promising target for the development of therapeutics to treat the disease and has been subject of extensive structural studies. Elaborate experiments suggested formation of a long-lived covalent intermediate in the catalytic mechanism and identified a Tyr/Glu pair as an unusual catalytic couple. This requires that the tyrosine hydroxyl proton is transferred to the carboxylate group of glutamate before the nucleophilic attack. Since the solution pK(a)s of tyrosine and glutamate are very different, this transfer can only be accomplished if the reaction environment selectively stabilizes the product state. We compute the free energy profile for the proton transfer in different environments, and our results indicate that it can take place in the active site of trans-sialidase, but only after substrate binding. By means of the energy decomposition method, we explain the influence that the active site residues exert on the reaction and how the pattern is changed when the substrate is present. This study represents an initial step that can shed light on our understanding of the catalytic mechanism of this reaction.     

Dynamics of proton transfer and enzymatic activity

The rate-determining elementary reaction step, i.e. proton transfer from the chymotrypsin active centre to the scissile substrate bond has been studied in the present work. On the basis of our theoretical results a hypothesis was formulated to explain chymotrypsin enzymatic efficiency. After ES complex formation excited vibrational states are populated in the enzyme molecule. In the rate-determining elementary reaction step, the proton transfer takes place from the first excited vibrational state of the N-H bond in the imidazole group of His57. This proton transfer is realised by quantum mechanical tunneling mechanism.