Substrate chirality and specificity of diacylglycerol kinases and the multisubstrate lipid kinase

The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times more rapidly than 2,3-dioleoylglycerol. The alpha and zeta isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective epsilon isoform. The inhibition by 2,3-dioleoylglycerol is uncompetitive. This corresponds to a kinetic scheme in which the inhibitor can bind to the enzyme-substrate complex, but not to the free enzyme. Our data indicate that despite their similar structures, 1,2-dioleoylglycerol and 2,3-dioleoylglycerol do not compete for the active site of these three isoforms of diacylglycerol kinase. We suggest that the 2,3-dioleoylglycerol binds to a site on the alpha and zeta isoforms of diacylglycerol kinase that is exposed as a consequence of the substrate binding to the active site. The chiral specificity of these enzymes thus mimics the substrate specificity, with MuLK being the least selective and the epsilon isoform of diacylglycerol kinase exhibiting the greatest selectivity.     

Structural basis for the substrate specificity of 3-hydroxybutyrate dehydrogenase

The substrate specificity of 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-hydroxybutyrate dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner.     

X-ray crystal structures of D100E trichodiene synthase and its pyrophosphate complex reveal the basis for terpene product diversity

The 2.4 A resolution X-ray crystal structure of D100E trichodiene synthase and the 2.6 A resolution structure of its complex with inorganic pyrophosphate are reported. The D100E amino acid substitution in the so-called "aspartate-rich" motif does not result in large changes to the overall structure of the enzyme. In the pyrophosphate complex, however, pyrophosphate coordinates two Mg(2+) ions at the mouth of the active site without causing large changes in the structure of the enzyme. This contrasts with pyrophosphate binding in the wild-type enzyme, where pyrophosphate coordinates three Mg(2+) ions and triggers a significant conformational change that closes the mouth of the active site and optimizes packing density in the enzyme-substrate complex. The attenuation of active site closure in D100E trichodiene synthase compromises enzyme-substrate packing density and confers additional spatial and conformational degrees of freedom on the substrate and carbocation intermediates, which in turn results in the formation of five alternate sesquiterpene products in addition to trichodiene. By extension, then, the diversity of terpene cyclases in biology may have evolved in part by amino acid substitutions that fine-tune structural changes dependent on metal-diphosphate complexation that govern the formation of the active site template and enzyme-substrate packing density.     

Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus

Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 A resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.     

Simulation of the substrate cavity dynamics of quercetinase

Molecular dynamics (MD) simulations have been performed on quercetin 2,3 dioxygenase (2,3QD) to study the mobility and flexibility of the substrate cavity. 2,3QD is the only firmly established Cu-containing dioxygenase known so far. It catalyses the breakage of the O-heterocycle of flavonols. The substrates occupy a shallow and overall hydrophobic cavity proximal to the metal centre of the homo-dimeric enzyme. The linker connecting the C-terminal and N-terminal domains in the monomer is partly disordered in the crystal structure and part of it forms a flexible lid at the entrance of the substrate cavity. This loop has been tentatively assigned a role in the enzyme mechanism: it helps lock the substrate into place. The dynamics of this loop has been investigated by MD simulation. The initial coordinates were taken from the crystal structure of 2,3QD in the presence of the substrate kaempferol (KMP). After equilibration and simulation over 7.2ns the substrate was removed and another equilibration and simulation of 7.2ns was performed. The results show that the structures of the free enzyme as well as of the enzyme-substrate complex are stable in MD simulation. The linker shows strongly enhanced mobility in the loop region that is close to the entrance to the substrate cavity (residues 154-169). Movement of the loop takes place on a timescale of 5-10ns. To confirm the conclusions about the loop dynamics drawn from the 7.2ns simulation, the simulation was extended with another 8ns. When substrate binds into the cavity the loop orders remarkably, although mobility is retained by residues 155-158. Some regions of the loop (residues 154-160 and 164-176) move over a considerable distance and approach the substrate closely, reinforcing the idea that they lock the substrate in the substrate cavity. The enthalpic component of the interaction of the loop with the protein and the KMP appears to favour the locking of the substrate. Two water molecules were found immobilised in the cavity, one of which exhibited rotation on the picosecond timescale. When the substrate is removed, the empty cavity fills up with water within 200ps.     

Molecular modeling of formate dehydrogenase: the formation of the Michaelis complex

The formation of the reactive enzyme-substrate complex of formate dehydrogenase has been investigated by molecular dynamics techniques accounting for different conformational states of the enzyme. Simulations revealed that the transport of substrate to the active site through the substrate channel proceeds in the open conformation of enzyme due to the crucial role of the Arg284 residue acting as a vehicle. However, formate binding in the active site of the open conformation leads to the formation of a nonproductive enzyme-substrate complex. The productive Michaelis complex is formed only in the closed enzyme conformation after the substrate and coenzyme have bound, when required rigidity of the binding site and reactive formate orientation due to interactions with Arg284, Asn146, Ile122, and His332 residues is attained. Then, the high occupancy (up to 75%) of the reactive substrate-coenzyme conformation is reached, which was demonstrated by hybrid quantum mechanics/molecular mechanics simulations using various semiempirical Hamiltonians.     

Functional analysis of the copper-dependent quercetin 2,3-dioxygenase. 2. X-ray absorption studies of native enzyme and anaerobic complexes with the substrates quercetin and myricetin

Quercetin 2,3-dioxygenase (2,3QD) is a mononuclear copper-dependent dioxygenase which catalyzes the cleavage of the heterocyclic ring of the flavonol quercetin (5,7,3',4'-tetrahydroxy flavonol) to produce 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide. In this study, X-ray absorption spectroscopy has been used to characterize the local structural environment of the Cu(2+) center of Aspergillus japonicus 2,3QD. Analysis of the EXAFS region of native 2,3QD at functionally relevant pH (pH 6.0) indicates an active site equally well-described by either four or five ligands (3N(His) + 1-2O) at an average distance of 2.00 A. Bond valence sum analysis confirms that the best model is somewhere between the two. When, however, 2,3QD is anaerobically complexed with its natural substrate quercetin, the copper environment undergoes a transition to a five-coordinated cage, which is also best modeled by a single shell of N/O scatterers at the average distance of 2.00 A. This coordination is independently confirmed by the anaerobic complex with myricetin (5'-hydroxy quercetin). XANES analysis confirms that substrate binding does not reduce the Cu(2+) ion. The present study gives the first direct insights into the coordination chemistry of the enzyme complexed with its substrates. It suggests that activation for O(2) attack is achieved by monodentate substrate complexation to the copper ion through the 3-hydroxyl group. In addition, monodentate carboxylate ligation by the Glu73 side chain is likely to play a role in the fine-tuning of the equilibrium leading to the formation of the activated E.S complex.     

Brownian dynamics simulation of substrate motion near active site of enzyme entrapped inside reverse micelle

Brownian dynamics simulation has been applied to analyze the influence of the electrostatic field of a reverse micelle on the enzyme-substrate complex formation inside a micelle. The probability that the enzyme-substrate complex will form from serine protease (trypsin) and the specific hydrophilic cationic substrate Nα-benzoyl-l-arginine ethyl ester has been studied within the framework of the encounter complex formation theory. It has been shown that surfactant charge, dipole moments created by charged surfactant molecules and counterions, and permittivity of the inner core of reverse micelles can all be used as regulatory parameters to alter the substrate orientation near the active site of the enzyme and to change the probability that the enzyme-substrate complex will form.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Purine nucleoside metabolism in the archaeon Pyrococcus furiosus is catalyzed by purine nucleoside phosphorylase (PfPNP) and 5'-deoxy-5'-methylthioadenosine phosphorylase (PfMTAP). These enzymes, characterized by 50% amino acid sequence identity, show non-common features of thermophilicity and thermostability and are stabilized by intramolecular disulfide bonds. PfPNP is highly specific for 6-oxopurine nucleosides while PfMTAP is characterized by a broad substrate specificity with 6-aminopurine nucleosides as preferred substrates. Amino acid sequence comparison clearly shows that the hypothetical active sites of PfPNP and PfMTAP are almost identical and that, in analogy with human 5'-deoxy-5'-methylthioadenosine phosphorylase and human purine nucleoside phosphorylase, residue changes at level of the same crucial positions could be responsible for the switch of substrate specificity. To validate this hypothesis we changed the putative active site of PfPNP by site-directed mutagenesis. Substrate specificity and catalytic efficiency of PfPNP mutants were then analyzed by kinetic studies and compared with the wild-type enzyme. We carried out the molecular modeling of PfPNP and PfMTAP to obtain a picture of the overall enzyme structure and to identify structural features as well as interactions playing critical roles in thermostability. Finally, we utilized the structural models of mutant enzyme-substrate complex to rationalize the functional effects of the mutations.     

An internal docking site stabilizes substrate binding to γ-secretase: Analysis by molecular dynamics simulations

Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid β (Aβ) peptides of different lengths. Longer Aβ peptides are associated with the formation of neurotoxic plaques related to Alzheimer’s disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aβ fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.     

Evidence for an essential arginine residue in the substrate binding site of the mammalian succinate dehydrogenase

Phenylglyoxal and 2,3-butanedione rapidly inactivate membrane-bound or soluble bovine heart succinate dehydrogenase. The inhibition of the enzyme by these reagents is completely prevented by saturating concentration of malonate. The modification of the active site sulfhydryl group by p-chloromercuribenzoate decreases the rate of the enzyme inhibition by phenylglyoxal and abolishes the protective effect of malonate. Kinetic data suggest that the inactivation by phenylglyoxal results from the modification of an essential arginine residue(s) which interacts with dicarboxylate to form the primary enzyme-substrate complex.     

The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.     

Mutation of Gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended alpha-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short alpha-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this alpha-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.     

Heparin/Heparan Sulfate 6-O-Sulfatase from Flavobacterium heparinum: INTEGRATED STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF ENZYME ACTIVE SITE AND SUBSTRATE SPECIFICITY*

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.     

Glycyl-tRNA synthetase uses a negatively charged pit for specific recognition and activation of glycine

The crystal structures of glycyl-tRNA synthetase (GlyRS) from Thermus thermophilus, a homodimeric class II enzyme, were determined in the enzyme-substrate and enzyme-product states corresponding to the first step of aminoacylation. GlyRS was cocrystallized with glycine and ATP, which were transformed by the enzyme into glycyl-adenylate and thus gave the enzyme-product complex. To trap the enzyme-substrate complex, the enzyme was combined with the glycine analog ethanolamine and ATP. The ligands are bound in fixed orientations in the substrate-binding pocket of the N-terminal active site domain, which contains the classical class II aminoacyl-tRNA synthetase (aaRS) fold. Since glycine does not possess a side-chain, much of the specificity of the enzyme is directed toward excluding any additional atoms beyond the alpha-carbon atom. Several carboxylate residues of GlyRS line the glycine binding pocket; two of them interact directly with the alpha-ammonium group. In addition, the enzyme utilizes the acidic character of the pro-L alpha-hydrogen atom by contacting it via a glutamate carboxylic oxygen atom. A guanidino eta-nitrogen atom of the class II aaRS-conserved motif 2 arginine interacts with the substrate carbonyl oxygen atom. These features serve to attract the small amino acid substrate into the active site and to position it in the correct orientation. GlyRS uses class II-conserved residues to interact with the ATP and the adenosine-phosphate moiety of glycyl-adenylate. On the basis of this similarity, we propose that GlyRS utilizes the same general mechanism as that employed by other class II aminoacyl-tRNA synthetases.     

Crystal structure and mechanism of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis

The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 A. TDO catalyzes the irreversible oxidation of l-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate l-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.     

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.     

Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize gamma-(32)P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.     

Catalytic properties of alkaline phosphatase from pig kidney

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.