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At least 343 records · Page 19Linked to original sources
PubMed2000

Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis.

BACKGROUND: Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are well-recognized antiparasitic drug targets. HGPRT is also a paradigmatic representative of the phosphoribosyltransferase family of enzymes, which includes other important biosynthetic and salvage enzymes and drug targets. To better understand the reaction mechanism of this enzyme, we have crystallized HGPRT from the apicomplexan protozoan Toxoplasma gondii as a ternary complex with a substrate and a substrate analog. RESULTS: The crystal structure of T. gondii HGPRT with the substrate Mg2+-PRPP and a nonreactive substrate analog, 9-deazaguanine, bound in the active site has been determined at 1.05 A resolution and refined to a free R factor of 15.4%. This structure constitutes the first atomic-resolution structure of both a phosphoribosyltransferase and the central metabolic substrate PRPP. This pre-transition state complex provides a clearer understanding of the structural basis for catalysis by HGPRT. CONCLUSIONS: Three types of substrate deformation, chief among them an unexpected C2'-endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-pi interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.

Animals↗
PubMed1999

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase.

BACKGROUND: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B12) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt-carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an alpha2beta2gamma2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase-cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. RESULTS: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers (alphabetagamma)2. The cobalamin molecule is bound between the alpha and beta subunits in the 'base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The alpha subunit includes a (beta/alpha)8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. CONCLUSIONS: This is the first crystallographic indication of the 'base-on' mode of cobalamin binding. An unusually long cobalt-base bond seems to favor homolytic cleavage of the cobalt-carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.

Amino Acid Sequence↗
PubMed2002

Rescue of an abasic hairpin ribozyme by cationic nucleobases: evidence for a novel mechanism of RNA catalysis.

The hairpin ribozyme catalyzes a reversible phosphodiester cleavage reaction. We examined the roles of conserved nucleobases in catalysis using an abasic ribozyme rescue strategy. Loss of the active site G8 nucleobase reduced the cleavage rate constant by 350-fold while loss of A9 and A10 nucleobases reduced activity less than 10-fold. Certain heterocyclic amines restored partial activity when provided in solution to the variant lacking G8. Heterocyclic amines that were capable of rescue shared the exocyclic amine and cyclic amide in common with the Watson-Crick hydrogen bonding face of guanine. In contrast to the shallow pH dependence of unmodified ribozyme activity, rescue activity increased sharply with decreasing pH. These results support a novel model for RNA catalysis in which a cationic nucleobase contributes electrostatic stabilization to negative charge developing in the transition state.

Base Sequence↗
PubMed1998

Interfacial catalysis by phospholipases at conjugated lipid vesicles: colorimetric detection and NMR spectroscopy.

BACKGROUND: Self-assembled conjugated polymers are rapidly finding biological and biotechnological applications. This work describes a synthetic membrane system based on self-assembled polydiacetylenes, which are responsive to the enzymatic activity of phospholipases - a ubiquitous class of enzymes that catalyze the hydrolysis of phospholipid molecules embedded in cell membranes. RESULTS: We show that phospholipases are active at bilayer vesicles composed of the natural enzyme substrate, dimyristoylphosphatidylcholine (DMPC), and a synthetic pi-conjugated polymerized lipid based on polydiacetylene (PDA). In addition, the enzymatic reaction induces an optical transition in the surrounding PDA matrix, visible to the naked eye. Nuclear magnetic resonance spectroscopy confirms the occurrence of enzymatic catalysis and reveals the fate of the cleavage products. CONCLUSIONS: The results indicate that the structural and color changes of the PDA matrix are directly related to interfacial catalysis by phospholipase. This novel biocatalytic method of inducing optical transitions in conjugated polymers might lead to new approaches towards rapidly screening new enzyme inhibitor compounds.

Acetylene↗
PubMed2000

Crystal structure of yeast Esa1 suggests a unified mechanism for catalysis and substrate binding by histone acetyltransferases.

Esa1 is the catalytic subunit of the NuA4 histone acetylase (HAT) complex that acetylates histone H4, and it is a member of the MYST family of HAT proteins that includes the MOZ oncoprotein and the HIV-1 Tat interacting protein Tip60. Here we report the X-ray crystal structure of the HAT domain of Esa1 bound to coenzyme A and investigate the protein's catalytic mechanism. Our data reveal that Esa1 contains a central core domain harboring a putative catalytic base, and flanking domains that are implicated in histone binding. Comparisons with the Gcn5/PCAF and Hat1 proteins suggest a unified mechanism of catalysis and histone binding by HAT proteins, whereby a structurally conserved core domain mediates catalysis, and sequence variability within a structurally related N- and C-terminal scaffold determines substrate specificity.

Acetyltransferases↗
PubMed2002

Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.

The catalytic mechanism of peptide bond formation on the ribosome is not known. The crystal structure of 50S ribosomal subunits shows that the catalytic center consists of RNA only and suggests potential catalytic residues. Here we report rapid kinetics of the peptidyl transferase reaction with puromycin at rates up to 50 s(-1). The rate-pH profile of the reaction reveals that protonation of a single ribosomal residue (pK(a) = 7.5), in addition to protonation of the nucleophilic amino group, strongly inhibits the reaction (>100-fold). The A2451U mutation within the peptidyl transferase center has about the same inhibitory effect. These results suggest a contribution to overall catalysis of general acid-base and/or conformational catalysis involving an ionizing group at the active site.

Binding Sites↗
PubMed2003

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis.

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.

Animals↗
PubMed2002

Core and periphery functionalized dendrimers for transition metal catalysis; a covalent and a non-covalent approach.

Dendrimers are well-defined hyperbranched macromolecules with characteristic globular structures for the larger systems. The recent impressive strides in synthetic procedures increased the accessibility of functionalized dendrimers at a practicable scale, resulting in a rapid development of dendrimer chemistry. Dendrimers have inspired many chemists to develop new materials and several applications have been explored, catalysis being one of them. The position of the catalytic site(s) as well as the spatial separation of the catalysts within the dendritic framework is of crucial importance. Dendrimers that are functionalized with transition metals in the core can potentially mimic properties of enzymes, their efficient natural counterparts, whereas the surface-functionalized systems have been proposed to fill the gap between homogeneous and heterogeneous catalysis. We prepared both core- and periphery-functionalized dendritic catalysts that are sufficiently large to enable separation by modern nanofiltration techniques. Here we review our recent findings using these promising novel transition metal-functionalized dendrimers as catalysts in several reactions. We will discuss some of the consequences of the architecturally different systems that have been studied and will elaborate on a novel non-covalent strategy of dendrimer functionalization.

Alkylation↗
PubMed2003

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors.

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.

Catalysis↗
PubMed2003

Catching catalysis in the act: using single crystal kinetics to trap methylamine dehydrogenase reaction intermediates.

Methylamine dehydrogenase (MADH) is produced by a range of gram-negative methylotrophic and autotrophic bacteria, and allows the organisms to utilise methylamine as the sole source of carbon. The enzyme catalyses the oxidation of methylamine to formaldehyde and ammonia, leaving it in a two-electron reduced state. To complete the catalytic cycle, MADH is reoxidised via an electron transfer (ET) chain. The redox center in the enzyme is the organic cofactor tryptophan tryptophylquinone (TTQ) derived from the posttranslational modification of two Trp residues in the protein. This cofactor has spectral features in the visible region, which change during catalytic turnover, defining spectrally distinct reaction intermediates that reflect the electronic state of the TTQ. In the case of the Paracoccus denitrificans enzyme the physiologic ET chain involves the protein redox partner amicyanin (a blue copper protein). A stable binary (MADH/amicyanin) complex can be formed, and its crystal structure has been solved to 2.5 A resolution by Chen et al. [Biochemistry 21 (1992) 4959]. These crystals were shown to be competent for catalysis and ET by Merli et al. [J. Biol. Chem. 271 (1996) 9177] using single crystal polarised absorption spectroscopy. Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze-trapping, we have trapped reaction intermediates of the enzyme in complex with its physiological redox partner amicyanin in the crystalline state. We will present data confirming that catalysis and ET in the binary complex crystals can be tracked by single crystal visible microspectrophotometry. We will also show that the reaction pathway is unperturbed by the presence of cryoprotectant solution, enabling direct freeze-trapping of reaction intermediates within the crystal. We will present new data demonstrating that the binary complex crystals are also capable of exhibiting UV light-dependent oxidase activity, as observed in solution [Biochim. Biophys. Acta 1364 (1998) 297].

Bacterial Proteins↗
PubMed2001

Elucidating the nature of enzyme catalysis utilizing a new twist on an old methodology: quantum mechanical-free energy calculations on chemical reactions in enzymes and in aqueous solution.

How do enzymes achieve very large rate enhancements compared to corresponding uncatalyzed reactions in solution? We present a computational approach which combines high-level ab initio quantum mechanical calculations with classical free energy calculations to address this question. Our calculations lead to accurate estimates of DeltaG for both trypsin and catechol O-methyltransferase-catalyzed and reference uncatalyzed reactions and give new insights into the nature of enzyme catalysis. The same methodology applied to steps in the catalytic mechanism of citrate synthase further supports the conclusion that one need not invoke special concepts such as "low-barrier hydrogen bonds" or "pK(a) matching" to explain enzyme catalysis.

Catalysis↗
PubMed2000

Synthetic catalysis of amide isomerization.

Rotation about the C-N bond in amides can be catalyzed by Bronsted and Lewis acids, as well as nucleophiles and bases. Catalysis of amide isomerization occurs in biological systems via "rotamase" enzymes; however, the mechanisms by which these proteins operate are not completely understood. We outline investigations that provide experimental support for mechanisms believed to be feasible for the catalysis of amide isomerization and present practical applications that have resulted from this work.

Amides↗
PubMedDate not supplied

Enzymes by design: chemogenetic assembly of transamination active sites containing lysine residues for covalent catalysis.

Artificial enzymes can be created by covalent conjugation of a catalytic active group to a protein scaffold. Here, two transamination catalysts were designed via computer modeling and assembled by chemically conjugating a pyridoxamine moiety within the large cavity of intestinal fatty acid binding protein. Each catalyst included a lysine residue, introduced via site-directed mutagenesis, that promotes catalysis by covalent interactions with the pyridoxamine group. Evidence for such interactions include the formation of a Schiff base with the pyridoxal form of the catalyst and a rate versus pH dependence that is bell shaped; both of these features are manifested in natural transaminases. The resulting constructs operate with high enantioselectivity (83-94% ee) and increase the rate of reaction as much as 4200-fold over the rate in the absence of the protein; this is a modest (12-fold) increase in catalytic efficiency (kcat/KM) compared to the conjugate lacking the lysine residue. Most importantly, these artificial aminotransferases are the first examples of designed bioconjugates capable of covalent catalysis, highlighting the potential of this chemogenetic approach.

Carrier Proteins↗
PubMed1995

Catalysis by hamster dihydroorotase: zinc binding, site-directed mutagenesis, and interaction with inhibitors.

Hamster dihydroorotase is the central domain of a trifunctional protein which has been cloned, overexpressed, and purified from Escherichia coli. Using the cDNA encoding the dihydroorotase domain, site-directed mutagenesis of amino acid residues conserved between species has enabled identification of three ligands of zinc at the catalytic site as His15, 17 and 158. The underlined amino acids of the nonapeptide sequence Ile12-Asp13-Val14-His15-Val16-His17- Leu18-Arg19-Glu20 from hamster are conserved between dihydroorotases from 8 species. It is proposed that the residues Asp13-His15-->ZnII form a triad at the active site and that Arg19, for which even the conservative mutation Arg19-->Lys yields an inactive enzyme, is involved in substrate binding. Site-directed mutagenesis of the conserved His186-->Ala yielded a mutant enzyme with a reduced affinity for 65Zn2+. The Km for dihydroorotate (DHO) increased from 4.0 to 11 microM, while the Vmax decreased from 1.2 to 0.53 mumol min-1 (mg of protein)-1, implicating this residue in only a minor way with binding of DHO and in catalysis. The mutation Asp230-->Glu resulted in a 14-fold increase in Km and a 16-fold decrease in Vmax, indicating involvement of this conserved residue in both binding and catalysis. The mutation Lys239-->Gly increased the Km for DHO 110-fold with a 2-fold increase in Vmax, suggesting that this residue may form a hydrogen bond with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Interfacial catalysis by phosphoinositide 3'-hydroxykinase.

Phosphorylation of phosphoinositides by phosphoinositide 3'-hydroxykinase (PI3K) occurs at a lipid/water interface. We have determined that highly purified recombinant human P13K binds tightly to vesicle interfaces composed primarily of phosphatidylinositol (PI) or 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM). The rate of desorption of PI3K from the vesicle interface is slow and does not significantly affect the observed product formation kinetics. Observations which demonstrate that PI3K is tightly bound to the vesicle lipid/water interface include the following: (1) product formation plateaus rapidly, even in the presence of active enzyme and excess substrate; (2) total product formation is proportional to the amount of PI3K; (3) initial product formation rates are unaffected by bulk lipid concentration but are dependent on the interfacial substrate concentration; and (4) PI3K partitions with lipid vesicles in sedimentation gradients. This enzymatic profile has been referred to as catalysis in the "scooting" mode (Berg et al., 1991). A kinetic analysis of PI3K catalysis in the scooting mode is presented. The interfacial Km,app for PI was determined to be approximately 6.0 mol % in PI/DMPM vesicles. The ratio of specificity constants (kcat/Km) for PI, phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-diphosphate (PIP2) utilization was determined to be near unity. These results provide a rigorous enzymological framework for the kinetic analysis of PI3K inhibitors.

Adenosine Triphosphate↗
PubMed1993

Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis.

Amino acid sequence comparisons reveal that tyrosine-152 and lysine-156 of Drosophila alcohol dehydrogenase (ADH) are conserved in homologous dehydrogenases, suggesting that these residues are important in catalysis. To test this hypothesis, we used site-directed mutagenesis to substitute tyrosine-152 with phenylalanine, histidine, or glutamic acid or to substitute lysine-156 with isoleucine. All of these mutants are catalytically inactive. Two mutants were active: A cysteine mutation of tyrosine-152 has 0.25% of wild-type ADH activity, while an arginine substitution of lysine-156 retains 2.2% of wild-type ADH activity. Kinetic analysis shows that the cysteine mutant increases Km(ethanol) 56-fold and Km(propan-2-ol) 100-fold, while Km(NAD) values are essentially unaltered. The arginine mutant also shows the significant enlargement of Km(ethanol), but not of Km(NAD). Furthermore, the cysteine mutant and arginine mutant have different substrate specificity and behave differently on competitive inhibition than wild-type ADH. These results suggest that both tyrosine-152 and lysine-156 have essential roles in catalysis by Drosophila ADH.

1-Propanol↗
PubMed1993

Use of binding energy in catalysis: optimization of rate in a multistep reaction.

The role of binding energy in optimizing the overall rate of catalysis by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been investigated by measuring the rate constants for transfer of tyrosine from engineered mutants to tRNA. The residues chosen for mutation are those that were previously identified as binding tyrosyl adenylate and contributing to the rate constant for its formation. It was previously found that tighter binding of the tyrosyl adenylate was accompanied by an increase in the rate constant for its formation. A new linear free energy relationship is presented that links the two. We now find that the rate constant for transfer of Tyr from Tyr-AMP to tRNA decreases with increasing stability of the E.Tyr-AMP complex on mutation of Thr-51. Position 51 is the one that is found to be the most variable of the binding-site residues among the enzymes from different species. The tightness of binding of the intermediate is thus a compromise, since stabilizing the intermediate speeds up the first step but slows down the second. The rate constants for activation and transfer by wild-type enzymes are very similar, which is optimal for the rate of the overall reaction. Those for the mutants diverge so that the rate of overall catalysis is lower.

Adenosine Monophosphate↗
PubMed1993

Kinetic analysis of NAD(+)-isocitrate dehydrogenase with altered isocitrate binding sites: contribution of IDH1 and IDH2 subunits to regulation and catalysis.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is an allosterically regulated enzyme that exists as an octamer composed of two nonidentical subunits, designated IDH1 and IDH2. To determine the contribution of each subunit to regulation and catalysis, a conserved serine residue at the proposed active site of each subunit was mutated to alanine. This mutation in IDH1 resulted in a 6-fold decrease in Vmax and a decrease in cooperativity, but little change in S0.5 for isocitrate. The mutant IDH2, in contrast, exhibited a 60-fold decrease in maximal velocity and a 2-fold reduction in S0.5 for isocitrate, but the cooperativity was unaffected. Responses to the allosteric modifier AMP also differed for the two mutant enzymes. The IDH1 mutant enzyme was not activated by AMP, whereas the IDH2 mutant enzyme exhibited an increase in isocitrate affinity in the presence of AMP similar to that observed with the wild-type enzyme. On the basis of these kinetic results, a model is presented which proposes that IDH1 functions as a regulatory subunit while IDH2 functions in catalysis. To determine if IDH1 or IDH2 alone is catalytically active, we also expressed the individual subunits in yeast strains in which the gene encoding the other subunit had been disrupted. Mitochondrial extracts from strains overexpressing solely IDH1 or IDH2 contained no detectable activity in the presence or absence of AMP. Gel filtration of these extracts showed that both IDH1 and IDH2 behaved as monomers, suggesting that the major subunit interactions within the octamer are between IDH1 and IDH2.

Adenosine Monophosphate↗