Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.     

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae

beta-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (beta-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nepsilon stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nepsilon of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.     

Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.     

Divergent evolution of the thiolase superfamily and chalcone synthase family

Enzymes of the thiolase superfamily catalyze the formation of carbon-carbon bond via the Claisen condensation reaction. Thiolases catalyze the reversible non-decarboxylative condensation of acetoacetyl-CoA from two molecules of acetyl-CoA, and possess a conserved Cys-His catalytic diad. Elongation enzymes (beta-ketoacyl-acyl carrier protein synthase (KAS) I and KAS II and the condensing domain of polyketide synthase) have invariant Cys and two His residues (CHH triad), while a Cys-His-Asn (CHN) triad is found in initiation enzymes (KAS III, 3-ketoacyl-CoA synthase (KCS) and the chalcone synthase (CHS) family). These enzymes all catalyze decarboxylative condensation reactions. 3-Hydroxyl-3-methylglutaryl-CoA synthase (HMGS) also contains the CHN triad, although it catalyzes a non-decarboxylative condensation. That the enzymes of the thiolase superfamily share overall similarity in protein structure and function suggested a common evolutionary origin. All thiolases were found to have, in addition to the Cys-His diad, either Asn or His (thus C(N/H)H) at a position corresponding to the His in the CHH and CHN triads. In our phylogenetic analyses, the thiolase superfamily was divided into four main clusters according to active site architecture. During the functional divergence of the superfamily, the active architecture was suggested to evolve from the C(H)H in archaeal thiolases to the C(N/H)H in non-archaeal thiolases, and subsequently to the CHH in the elongation enzymes and the CHN in the initiation enzymes. Based on these observations and available biochemical and structural evidences, a plausible evolutionary history for the thiolase superfamily is proposed that includes the emergence of decarboxylative condensing enzymes accompanied by a recruitment of the His in the CHH and CHN triads for a catalytic role during decarboxylative condensation. In addition, phylogenetic analysis of the plant CHS family showed separate clustering of CHS and non-CHS members of the family with a few exceptions, suggesting repeated gene birth-and-death and re-invention of non-CHS functions throughout the evolution of angiosperms. Based on these observations, predictions on the enzymatic functions are made for several members of the CHS family whose functions are yet to be characterized. Further, a moss CHS-like enzyme that is functionally similar to a cyanobacterial enzyme was identified as the most recent common ancestor to the plant CHS family.     

In vitro condensation of amino acids in aqueous media: A synthesis driven by catalytic carbon monoxide oxidation

A novel mechanism is proposed for amino acid condensations to oligopeptides in aqueous media. The palladium-catalyzed condensation appears to proceed through a mechanism involving the Pd(0)/Pd(II) redox cycle. The reaction requires the use of carbon monoxide and an oxidant, and it is proposed that the oxidation of carbon monoxide to carbon dioxide drives the otherwise thermodynamically uphill condensation. The reaction occurs for several amino acids including glycine, alanine, β-alanine, and 5-aminopentanoic acid. Competition studies between glycine and alanine reveal steric effects on product formation. Dipeptides are the predominant condensation products, though some longer chains, containing up to 4 amino acids, can be observed. First-order plots for dipeptide appearance show an inverse, secondary, isotope effect consistent with the rate determining carboxylic-carbamic anhydride formation.     

Hydrolysis of organophosphate compounds by mutant butyrylcholinesterase: a story of two histidines

This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds by G117H-BChE. It is a theoretical study that focuses on the role of the G117H mutation in the dephosphorylation step. Various proposed mechanisms are examined. We show that His117 acts as a general base by activating a water molecule, and thus assisting its nucleophilic attack on the phosphate. The calculated reaction energy profile agrees well with the experimental data. Moreover, analysis of the reaction via its two hypothetical elementary steps, proton transfer and hydroxide attack, supports the role of His117 as a general base. Further support to the proposed mechanism is gained by structural comparison of the active site to RNAse A, which has similar composition of substrate and functional groups. The similarity between these enzymes extends beyond the structure and also becomes evident when comparing functionality of various active sites residues as well as rate-pH dependence obtained in the two cases. Moreover, it is demonstrated that an extended form of Bevilacqua's model (Biochemistry 2003;42:2259-2265) may resolve the apparent contradictions between the proposed mechanism and various experimental observations regarding rate-pH dependence. Finally, that same model is shown to rationalize the hydrolase activity of G117D BChE, an observation which is considered puzzling. It is concluded that G117H-BChE hydrolyzes echothiophate and possibly other OP compounds via a general acid-base mechanism. On the basis of this mechanism, one can now proceed with rational design aimed at improving the enzyme by exploiting both the structural and mechanistic knowledge.     

The 1.8 A crystal structure and active-site architecture of beta-ketoacyl-acyl carrier protein synthase III (FabH) from escherichia coli

Background: beta-Ketoacyl-acyl carrier protein synthase III (FabH) initiates elongation in type II fatty acid synthase systems found in bacteria and plants. FabH is a ubiquitous component of the type II system and is positioned ideally in the pathway to control the production of fatty acids. The elucidation of the structure of FabH is important for the understanding of its regulation by feedback inhibition and its interaction with drugs. Although the structures of two related condensing enzymes are known, the roles of the active-site residues have not been experimentally tested. Results: The 1.8 A crystal structure of FabH was determined using a 12-site selenium multiwavelength anomalous dispersion experiment. The active site (Cys112, His244 and Asn274) is formed by the convergence of two alpha helices and is accessed via a narrow hydrophobic tunnel. Hydrogen-bonding networks that include two tightly bound water molecules fix the positions of His244 and Asn274, which are critical for the decarboxylation and condensation reactions. Surprisingly, the His244-->Ala mutation does not affect the transacylation reaction suggesting that His244 has only a minor influence on the nucleophilicity of Cys112. Conclusions: The histidine and asparagine active-site residues are both required for the decarboxylation step in the condensation reaction. The nucleophilicity of the active-site cysteine is enhanced by the alpha-helix dipole effect, and an oxyanion hole promotes the formation of the tetrahedral transition state.     

Swiveling domain mechanism in pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.     

Role of two histidines in the (6-4) photolyase reaction

The reaction mechanism of Xenopus (6-4) photolyase was investigated using several mutant enzymes. In the active site, which is homologous between the cis,syn-cyclobutane pyrimidine dimer and (6-4) photolyases, four amino acid residues that are specific to (6-4) photolyase, Gln(288), His(354), Leu(355), and His(358), and two conserved tryptophans, Trp(291) and Trp(398), were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp(291) and Trp(398) mutants retained some activity. Taking the pH profile of the (6-4) photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.     

High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.     

Reassessing the type I dehydroquinate dehydratase catalytic triad: Kinetic and structural studies of Glu86 mutants

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined—Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction—the role of Glu86 remains poorly characterized. To probe Glu86′s role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild‐type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.     

Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

The condensation step of fatty acid elongation is the addition of a C₂ unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.     

Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.     

Crystal structure of the yeast His6 enzyme suggests a reaction mechanism

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.     

Computational studies of proton transport through the M2 channel

The M2 ion channel is an essential component of the influenza A virus. This low-pH gated channel has a high selectivity for protons. Evidence from various experimental data has indicated that the essential structure responsible for the channel is a parallel homo-tetrameric alpha-helix bundle having a left-handed twist with each helix tilted with respect to the membrane normal. A backbone structure has been determined by solid state nuclear magnetic resonance (NMR). Though detailed structures for the side chains are not available yet, evidence has indicated that His37 and Trp41 in the alpha-helix are implicated in the local molecular structure responsible for the selectivity and channel gate. More definitive conformations for the two residues were recently suggested based on the known backbone structure and recently obtained NMR data. While two competitive proton-conductance mechanisms have been proposed, the actual proton-conductance mechanism remains an unsolved problem. Computer simulations of an excess proton in the channel and computational studies of the His37/Trp41 conformations have provided insights into these structural and mechanism issues.     

Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.     

Mechanistic enzymology of serine palmitoyltransferase

Serine palmitoyltransferase, which is one of the α-oxamine synthase family enzymes, catalyzes the condensation reaction of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine, the first and rate-determining step of the sphingolipid biosynthesis. As with other α-oxamine synthase family enzymes, the catalytic reaction is composed of multiple elementary steps, and the mechanism to control these steps to avoid side reactions has been the subject of intensive research in recent years. Combined spectroscopic, kinetic, and structural studies have revealed the finely controlled stereochemical mechanism, in which the His residue conserved among the α-oxamine synthase family enzymes plays a central and critical role. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

A theoretical study of the mechanism for peptide hydrolysis by thermolysin

The catalytic mechanism for peptide hydrolysis by thermolysin has been investigated using the B3LYP hybrid density functional method. The starting structure for the calculations was based on the X-ray crystal structure of the enzyme inhibited with the ZF (p)LA phosphonamidate transition-state analogue. Besides the three Zn ligands His142, His146 and Glu166, a few additional residues were also included in the model. Following the order of importance, the outer-sphere ligands Glu143, His231 and Asp226 were shown to play significant catalytic roles, well correlated with results from site-directed mutagenesis experiments. A single-step reaction mechanism was obtained starting from the initial enzyme-substrate complex with a pentacoordinated metal center and proceeding to the enzyme-carboxylate complex as a final product, following a proposal by Matthews and co-workers. The transition state combines a nucleophilic water oxygen attack on the peptide carbon and a proton transfer from the water to the peptide nitrogen, mediated by the Glu143 carboxylate. A free activation energy of 15.2 kcal/mol was obtained, compared to the experimental 12.4-16.3 kcal/mol range for various peptide substrates. An interesting aspect of the present single-step mechanism is that the Glu143 carboxylate moves a significant distance of ~1.0 A. Different chemical models were examined, both related to the system size and proper side-chain modeling. The significance of the protein frame rigidity around the active site was estimated by fixing and subsequently releasing the edge atom positions. Finally, alternative mechanistic proposals are briefly summarized.