NM23/NDP kinases play an important role in development and cancer
but their biological function is unknown, despite an intriguing
collection of biochemical properties including nucleoside-diphosphate
kinase (NDP kinase), DNA binding and transcription, a mutator function,
and cleavage of unusually structured DNA by means of a covalent
enzyme–DNA complex. To assess the role of the nuclease in human
NM23-H2, we sought to identify the amino acid responsible for covalent
catalysis. By sequencing a DNA-linked peptide and by site-directed
mutagenesis, we identified lysine-12, a phylogenetically conserved
residue, as the amino acid forming the covalent complex with DNA. In
particular, the ɛ-amino group acts as the critical nucleophile,
because substitution with glutamine but not arginine completely
abrogated covalent adduct formation and DNA cleavage, whereas the
DNA-binding properties remained intact. These findings and chemical
modification data suggest that phosphodiester-bond cleavage occurs by a
DNA glycosylase/lyase-like mechanism known as the signature of base
excision DNA repair nucleases. Involvement of NM23/NDP kinase in a
DNA repair pathway would be consistent with its role in normal and
tumor cell development. Additionally...
The GroE chaperones of Escherichia coli assist protein folding under physiological and heat shock conditions in an ATP-dependent way. Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved. Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions. We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way. GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold. Interestingly, catalysis of the folding reaction requires the formation of symmetrical football-shaped GroEL·GroES2 particles and the intermediate release of the nonnative protein from the chaperone complex. Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding.
Site-directed mutagenesis of selected residues of mammalian protein phosphatase 1 (PP-1) has been carried out to further define the mechanism of catalysis, activation by divalent cations, and inhibition by toxins and inhibitory proteins. Mutation of active site residues predicted to bind metals (N124D and H248N) resulted in a large loss of enzyme activity and decreased affinity for metal ions; mutation of residues predicted to bind phosphosubstrate (R96A or R221S) led to a large loss of enzyme activity; and mutation of active site residues (D95A and D208A) resulted in a large loss of enzyme activity. Mutants N124D, H248N, R96A, and R221S exhibited large decreases in sensitivity to the toxins calyculin A, okadaic acid, and microcystin and to thiophospho-DARPP-32. Mutation of Y272 (Y272F) had little effect on activity but resulted in a large decrease in sensitivity to okadaic acid and calyculin A. Mutant D208A exhibited a decrease in sensitivity to okadaic acid and calyculin A, but, paradoxically, the sensitivity to inhibition by thiophospho-DARPP-32 was increased. Mutation of acidic groove residues (E256R, E275R, E252A:D253A, and E252A:D253A:E256R) exhibited little change in enzyme activity and no change in sensitivity to toxins, but increased sensitivity to thiophospho-DARPP-32. These results suggest that toxins and phospho-DARPP-32 interact at the active site of PP-1 in a similar fashion despite their differences in structure. In addition...
We have studied the effect of the 3′ terminal CCA sequence in precursors of tRNAs on catalysis by the RNase P RNA or the holoenzyme from the cyanobacterium Synechocystis sp. PCC 6803 in a completely homologous system. We have found that the absence of the 3′ terminal CCA is not detrimental to activity, which is in sharp contrast to what is known in other bacterial systems. We have found that this is also true in other cyanobacteria. This situation correlates with the anomalous structure of the J15/16 loop in cyanobacteria, which is an important loop in the CCA interaction in Escherichia coli RNase P, and with the fact that cyanobacteria do not code the CCA sequence in the genome but add it posttranscriptionally. Modification of nucleotides 330–332 in the J15/16 loop of Synechocystis RNase P RNA from GGU to CCA has a modest effect on kcat for CCA-containing substrates and has no effect on cleavage-site selection. We have developed a direct physical assay of the interaction between RNase P RNA and its substrate, which was immobilized on a filter, and we have determined that Synechocystis RNase P RNA binds with better affinity the substrate lacking CCA than the substrate containing it. Our results indicate a mode of substrate binding in RNase P from cyanobacteria that is different from binding in other eubacteria and in which the 3′ terminal CCA is not involved.
3-hydroxy-3-methylglutaryl–CoA (HMG-CoA) reductase is the rate-limiting enzyme and the first committed step in the biosynthesis of cholesterol in mammals. We have determined the crystal structures of two nonproductive ternary complexes of HMG-CoA reductase, HMG-CoA/NAD+ and mevalonate/NADH, at 2.8 Å resolution. In the structure of the Pseudomonas mevalonii apoenzyme, the last 50 residues of the C terminus (the flap domain), including the catalytic residue His381, were not visible. The structures of the ternary complexes reported here reveal a substrate-induced closing of the flap domain that completes the active site and aligns the catalytic histidine proximal to the thioester of HMG-CoA. The structures also present evidence that Lys267 is critically involved in catalysis and provide insights into the catalytic mechanism.
In a previous examination using natural all-RNA substrates
that contained either a 5′-oxy or 5′-thio leaving group at the cleavage
site, we demonstrated that (i) the attack by the
2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only
for the substrate that contains a 5′-thio group (R11S) and
(ii) the departure of the 5′ leaving group is the
rate-limiting step for the natural all-RNA substrate (R11O) in both
nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy
diagrams for these reactions were provided in our previous publication.
In this report we found that the rate of cleavage of R11O by a
hammerhead ribozyme was enhanced 14-fold when Mg2+ ions
were replaced by Mn2+ ions, whereas the rate of cleavage of
R11S was enhanced only 2.2-fold when Mg2+ ions were
replaced by Mn2+ ions. This result appears to be exactly
the opposite of that predicted from the direct coordination of the
metal ion with the leaving 5′-oxygen, because a switch in metal ion
specificity was not observed with the 5′-thio substrate. However, our
quantitative analyses based on the previously provided energy diagram
indicate that this result is in accord with the double-metal-ion
mechanism of catalysis.
The equilibrium for formation of the intramolecular hydrogen bond
(KHB) in a series of substituted salicylate monoanions was
investigated as a function of ΔpKa, the difference
between the pKa values of the hydrogen bond donor and
acceptor, in both water and dimethyl sulfoxide. The dependence of log
KHB upon ΔpKa is linear in both solvents, but
is steeper in dimethyl sulfoxide (slope = 0.73) than in water
(slope = 0.05). Thus, hydrogen bond strength can undergo
substantially larger increases in nonaqueous media than aqueous
solutions as the charge density on the donor or acceptor atom
increases. These results support a general mechanism for enzymatic
catalysis, in which hydrogen bonding to a substrate is strengthened as
charge rearranges in going from the ground state to the transition
state; the strengthening of the hydrogen bond would be greater in a
nonaqueous enzymatic active site than in water, thus providing a rate
enhancement for an enzymatic reaction relative to the solution
reaction. We suggest that binding energy of an enzyme is used to fix
the substrate in the low-dielectric active site, where the
strengthening of the hydrogen bond in the course of a reaction is
The origin of the catalytic power of enzymes is discussed,
paying attention to evolutionary constraints. It is pointed out that
enzyme catalysis reflects energy contributions that cannot be
determined uniquely by current experimental approaches without
augmenting the analysis by computer simulation studies. The use of
energy considerations and computer simulations allows one to exclude
many of the popular proposals for the way enzymes work. It appears that
the standard approaches used by organic chemists to catalyze reactions
in solutions are not used by enzymes. This point is illustrated by
considering the desolvation hypothesis and showing that it cannot
account for a large increase in kcat relative to the
corresponding kcage for the reference reaction in a solvent
cage. The problems associated with other frequently invoked mechanisms
also are outlined. Furthermore, it is pointed out that mutation studies
are inconsistent with ground state destabilization mechanisms. After
considering factors that were not optimized by evolution, we review
computer simulation studies that reproduced the overall catalytic
effect of different enzymes. These studies pointed toward electrostatic
effects as the most important catalytic contributions. The nature of
this electrostatic stabilization mechanism is far from being obvious
because the electrostatic interaction between the reacting system and
the surrounding area is similar in enzymes and in solution. However...
The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-angstrom and 1.9-angstrom resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design.
A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone-labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected.
Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base...
During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli. In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F1. Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1. After reduction of the initial nonradioactive beta-gamma crosslink...
A tetramer of Mu transposase (MuA) cleaves the phage Mu DNA and joins these ends to a target DNA to catalyze transposition. Substitution mutations at Asp-269 or Glu-392 within MuA destroy both the DNA cleavage and joining activities without blocking tetramer assembly, indicating that the mutations specifically affect catalysis. Although inactive under standard reaction conditions (10 mM Mg2+), the mutant proteins are partially resuscitated by 10-20 mM Mn2+, concentrations 5- to 10-fold higher than optimal for wild-type MuA. Amino acid sequence alignment and the similar effects of mutations suggests that Asp-269 and Glu-392 of MuA may be analogs of the first Asp and final Glu of a conserved triad of acidic amino acids present in many transposases and the retroviral integrases (the D-D-35-E motif). The higher Mn2+ optima observed with MuA derivatives altered at these positions supports a role for the conserved acidic amino acids in coordinating divalent metal ions in the active sites of transposases.
Phosphotransacetylase (EC 126.96.36.199) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH3COOPO32− + CoASH ⇆ CH3COSCoA + HPO42−. The role of arginine residues was investigated for the phosphotransacetylase from Methanosarcina thermophila. Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3′-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3′-phosphate of CoA. Arg 133 is postulated to interact with the 5′-phosphate of CoA. Large decreases in kcat and kcat/Km for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases in kcat and kcat/Km were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2...
Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterpene precursor geranylgeranyl diphosphate to the stable bicyclic intermediate copalyl diphosphate. In the second, magnesium ion-dependent reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone and is coupled, by intramolecular proton transfer within a transient pimarenyl intermediate, to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane structure. Alternative deprotonations of the terminal abietenyl carbocation provide a mixture of abietadiene, levopimaradiene, and neoabietadiene, and this product profile varies as a function of pH. Mutational analysis of amino acids at the active site of a modeled structure has identified residues critical for catalysis, as well as several that play roles in specifying product formation, apparently by ligation of a magnesium ion cofactor. These results strongly suggest that choice between alternatives for deprotonation of the abietenyl intermediate depends more on the positioning effects of the carbocation–diphosphate anion reaction partners than on the pKa of multiple participating bases. In one extreme case...
Molecular dynamics studies of the Escherichia coli chorismate mutase (EcCM), containing at the active site chorismate and in turn the transition state (TS), have been performed. The simulations show that TS is not bound any tighter than chorismate. Comparison of average polar interactions show they are virtually identical for interactions of EcCM with chorismate and the TS, whereas hydrophobic interactions with TS are much weaker than with chorismate. Interactions and the mechanism of catalysis of chorismate → prephenate by the EcCM enzyme are discussed.
A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.
Nitric oxide (NO⋅) is a short-lived physiological messenger. Its various biological activities can be preserved in a more stable form of S-nitrosothiols (RS-NO). Here we demonstrate that at physiological NO⋅ concentrations, plasma albumin becomes saturated with NO⋅ and accelerates formation of low-molecular-weight (LMW) RS-NO in vitro and in vivo. The mechanism involves micellar catalysis of NO⋅ oxidation in the albumin hydrophobic core and specific transfer of NO+ to LMW thiols. Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating LMW thiols. Results suggest that the hydrophobic phase formed by albumin serves as a major reservoir of NO⋅ and its reactive oxides and controls the dynamics of NO⋅-dependant processes in the vasculature.
To exert control over RNA folding and catalysis, both molecular engineering strategies and in vitro selection techniques have been applied toward the development of allosteric ribozymes whose activities are regulated by the binding of specific effector molecules or ligands. We now describe the isolation and characterization of a new and considerably versatile RNA element that functions as a communication module to render disparate RNA folding domains interdependent. In contrast to some existing communication modules, the novel 9-nt RNA element is demonstrated to function similarly between a variety of catalysts that include the hepatitis delta virus, hammerhead, X motif and Tetrahymena group I ribozymes, and various ligand-binding domains. The data support a mechanistic model of RNA folding in which the element is comprised of both canonical and non-canonical base pairs and an unpaired nucleotide in the active, effector-bound conformation. Aside from enabling effector-controlled RNA function through rational design, the element can be utilized to identify sites in large RNAs that are susceptible to effector regulation.
The functional groups found among the RNA bases and in the phosphoribose backbone represent a limited repertoire from which to construct a ribozyme active site. This work investigates the possibility that simple RNA phosphodiester and hydroxyl functional groups could catalyze amide bond synthesis. Reaction of amine groups with activated esters would be catalyzed by a group that stabilizes the partial positive charge on the amine nucleophile in the transition state. 2′-Amine substitutions adjacent to 3′-phosphodiester or 3′-hydroxyl groups react efficiently with activated esters to form 2′-amide and peptide products. In contrast, analogs in which the 3′-phosphodiester is replaced by an uncharged phosphotriester or is constrained in a distal conformation react at least 100-fold more slowly. Similarly, a nucleoside in which the 3′-hydroxyl group is constrained trans to the 2′-amine is also unreactive. Catalysis of synthetic reactions by RNA phosphodiester and ribose hydroxyl groups is likely to be even greater in the context of a preorganized and solvent-excluding catalytic center. One such group is the 2′-hydroxyl of the ribosome-bound P-site adenosine substrate, which is close to the amine nucleophile in the peptidyl synthesis reaction. Given ubiquitous 2′-OH groups in RNA...