Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.     

Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.     

Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

Formamidopyrimidine-DNA-glycosylase (Fpg pro tein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2′-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation.     

Conformational changes in ammonia-channeling glutamine amidotransferases

Glutamine amidotransferases (GATs), which catalyze the synthesis of different aminated products, channel ammonia over 10-40 A from a glutamine substrate at the glutaminase site to an acceptor substrate at the synthase site. Ammonia production usually uses a cysteine-histidine-glutamate triad or a N-terminal cysteine residue. Crystal structures of several amidotransferase ligand complexes, mimicking intermediates along the catalytic cycle, have now been determined. In most cases, acceptor binding triggers glutaminase activation through domain-hinged movements and other conformational changes. Structural information shows how flexible loops of the synthase and glutaminase domains move to shield the two catalytic sites and anchor the substrates, and how the ammonia channel forms and opens or closes.     

Synchronous reversible alterations in enzymatic activity (conformational fluctuations) in actomyosin and creatine kinase preparations.

The phenomenon of synchronism of oscillations of actomyosin and creatine kinase activity in the whole volume of the enzyme preparations was analysed. The synchronous "conformational oscillations" were observed in concentrated gels of actomyosin and in diluted actomyosin and creatine kinase solutions (ATP-creatine N-phosphotransferase, EC 2.7.3.2). The macromolecules of proteins studied may be in two or four conformational states differing enzymatic activity. Large fluctuations become possible in a range of conditions wherein two or four different states, or conformers, are equiprobable. The synchronization of conformational changes of separate macromolecules is maintained with energy derived, for instance, from some oxidative process or dilution of the solution, the process being displayed as conformational oscillations.     

Conformational dynamics of abasic DNA upon interactions with AP endonuclease 1 revealed by stopped-flow fluorescence analysis

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.     

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.     

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.     

Modulation of individual steps in group I intron catalysis by a peripheral metal ion

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.     

Kinetic analysis of hydrolysis of free ATP and MgATP by natural actomyosin]

Computer analysis of experimental data published in 1-3 allowed to establish the presence of two non-interacting inequivalent hydrolytic sites in actomyosin molecule, one of them being specific for binding and hydrolysis of free ATP, the other--for MgATP. Thus both species of ATP are the substrates of actomyosin ATPase. Actomyosin molecule seems to bind on more (in additon to two active sites) substrate molecule (MgATP) at some non-catalytic regulatory site. The formation of the enzyme-substrate complex having three ATP molecules (one molecule of free ATP and two--of MgATP) is accompanied by the loss of the activity. An approach to the research of kinetic equations for complex systems considerably decreasing a number of variations to consider is given in this work.     

Cytomegalovirus capsid protease: biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin)

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na(2)SO(4) reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.     

Diverse role of conformational dynamics in carboxypeptidase A-driven peptide and ester hydrolyses: disclosing the "perfect induced fit" and "protein local unfolding" pathways by altering protein stability

Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of "initial rates" is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus "active" product) concentration, made it possible to rigorously determine the physically meaningful "effective" values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S(3) subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the "promoted water" mechanism for the peptide substrate that, seemingly, provides the almost "perfect induced fit" (low-barrier conformational adaptation), and (b) presumably, the "anhydride intermediate" mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, "partial or local unfolding") of the protein environment in the course of the rate-determining step.     

Two pockets in the active site of maize sesquiterpene synthase TPS4 carry out sequential parts of the reaction scheme resulting in multiple products

One of the most interesting features of terpene synthases is their ability to form multiple products with different carbon skeletons from a single prenyl diphosphate substrate. The maize sesquiterpene synthase TPS4, for example, produces a mixture of 14 different olefinic sesquiterpenes. To understand the complex TPS4 reaction mechanism, we modeled the active site cavity and conducted docking simulations with the substrate farnesyl diphosphate, several predicted carbocation intermediates, and the final reaction products. The model suggests that discrete steps of the reaction sequence are controlled by two different active site pockets, with the conformational change of the bisabolyl cation intermediate causing a shift from one pocket to the other. Site-directed mutagenesis and measurements of mutant activity in the presence of (E,E)- and (Z,E)-farnesyl diphosphate as substrates were employed to test this model. Amino acid alterations in pocket I indicated that early steps of the catalytic process up to the formation of the monocyclic bisabolyl cation are probably localized in this compartment. Mutations in pocket II primarily inhibited the formation of bicylic compounds, suggesting that secondary cyclizations of the bisabolyl cation are catalyzed in pocket II.     

Stochastic ensembles, conformationally adaptive teamwork, and enzymatic detoxification

It has been appreciated for a long time that enzymes exist as conformational ensembles throughout multiple stages of the reactions they catalyze, but there is renewed interest in the functional implications. The energy landscape that results from conformationlly diverse poteins is a complex surface with an energetic topography in multiple dimensions, even at the transition state(s) leading to product formation, and this represents a new paradigm. At the same time there has been renewed interest in conformational ensembles, a new paradigm concerning enzyme function has emerged, wherein catalytic promiscuity has clear biological advantages in some cases. "Useful", or biologically functional, promiscuity or the related behavior of "multifunctionality" can be found in the immune system, enzymatic detoxification, signal transduction, and the evolution of new function from an existing pool of folded protein scaffolds. Experimental evidence supports the widely held assumption that conformational heterogeneity promotes functional promiscuity. The common link between these coevolving paradigms is the inherent structural plasticity and conformational dynamics of proteins that, on one hand, lead to complex but evolutionarily selected energy landscapes and, on the other hand, promote functional promiscuity. Here we consider a logical extension of the overlap between these two nascent paradigms: functionally promiscuous and multifunctional enzymes such as detoxification enzymes are expected to have an ensemble landscape with more states accessible on multiple time scales than substrate specific enzymes. Two attributes of detoxification enzymes become important in the context of conformational ensembles: these enzymes metabolize multiple substrates, often in substrate mixtures, and they can form multiple products from a single substrate. These properties, combined with complex conformational landscapes, lead to the possibility of interesting time-dependent, or emergent, properties. Here we demonstrate these properties with kinetic simulations of nonequilibrium steady state (NESS) behavior resulting from energy landscapes expected for detoxification enzymes. Analogous scenarios with other promiscuous enzymes may be worthy of consideration.     

Intermediates and transition states in thiamin diphosphate-dependent decarboxylases. A kinetic and NMR study on wild-type indolepyruvate decarboxylase and variants using indolepyruvate, benzoylformate, and pyruvate as substrates

The thiamin diphosphate (ThDP)-dependent enzyme indolepyruvate decarboxylase (IPDC) is involved in the biosynthetic pathway of the phytohormone 3-indoleacetic acid and catalyzes the nonoxidative decarboxylation of 3-indolepyruvate to 3-indoleacetaldehyde and carbon dioxide. The steady-state distribution of covalent ThDP intermediates of IPDC reacting with 3-indolepyruvate and the alternative substrates benzoylformate and pyruvate has been analyzed by (1)H NMR spectroscopy. For the first time, we are able to isolate and directly assign covalent intermediates of ThDP with aromatic substrates. The intermediate analysis of IPDC variants is used to infer the involvement of active site side chains and functional groups of the cofactor in distinct catalytic steps during turnover of the different substrates. As a result, three residues (glutamate 468, aspartate 29, and histidine 115) positioned perpendicular to the thiazolium moiety of ThDP are involved in binding of all substrates and decarboxylation of the respective tetrahedral ThDP-substrate adducts. Most likely, interactions of these side chains with the substrate-derived carboxylate account for an optimal orientation of the substrate and/or intermediate in the course of carbon-carbon ligation and decarboxylation supporting the suggested least-motion, maximum overlap mechanism. The active site residue glutamine 383, which is located at the opposite site of the thiazolium nucleus as the "carboxylate pocket" (formed by the Glu-Asp-His triad), is central to the substrate specificity of IPDC, probably through orbital alignment. The Glu51-cofactor proton shuttle is, conjointly with the Glu-Asp-His triad, involved in multiple proton transfer steps, including ylide generation, substrate binding, and product release. Studies with para-substituted benzoylformate substrates demonstrate that the electronic properties of the substrate affect the stabilization or destabilization of the carbanion intermediate or carbanion-like transition state and in that way alter the rate dependence on decarboxylation. In conclusion, general mechanistic principles of catalysis of ThDP-dependent enzymes are discussed.     

The mechanism of action of mitochondrial ATPase (ATP synthase)

The F₁ part of the ATP synthase contains 6 nucleotide binding sites, four of which can be occupied and covalently labeled with 8-azido-adenine nucleotides. The other two sites contain tightly bound nucleotides that cannot be replaced by 8-azido-adenine nucleotides. Of the four exchangeable sites two are directly ivolved in catalysis and these are located on -subunits, while the other two are located at - interfaces and have probably a regulatory role by influencing the affinity of the catalytic sites for substrate and product. When only one catalytic site contains substrate the affinity is very high, the rate of hydrolysis is slow, and the dissociation of products is even slower (single-site catalysis). When the second site also becomes occupied, the affinity decreases enormously, and the rate of hydrolysis and dissociation of products increases several orders of magnitude. When, however, the second site is occupied by substrate in such a way that turnover is not possible at this site (e.g., covalent linkage of nitreno-ATP), the first site is no longer active, apart from the very slow single-site catalysis. The two nonexchangeable, tightly bound nucleotides that cannot be replaced by 8-azido-nucleotides, can be replaced by 2-azido-nucleotides, due to their anticonfiguration. This anticonfiguration of the substrate is also required for binding with high affinity to a catalytic site. A picture emerges in which one of the three - pairs of F₁ contains tightly bound, nonexchangeable nucleotides, while the other two contain both one catalytic site (on ) and one regulatory site (at the - interface). Cooperativity exists both between the two catalytic sites and between the catalytic and the regulatory sites.     

The structure of Ap(4)A hydrolase complexed with ATP-MgF(x) reveals the basis of substrate binding

Ap(4)A hydrolases are Nudix enzymes that regulate intracellular dinucleoside polyphosphate concentrations, implicating them in a range of biological events, including heat shock and metabolic stress. We have demonstrated that ATP x MgF(x) can be used to mimic substrates in the binding site of Ap(4)A hydrolase from Lupinus angustifolius and that, unlike previous substrate analogs, it is in slow exchange with the enzyme. The three-dimensional structure of the enzyme complexed with ATP x MgF(x) was solved and shows significant conformational changes. The substrate binding site of L. angustifolius Ap(4)A hydrolase differs markedly from the two previously published Nudix enzymes, ADP-ribose pyrophosphatase and MutT, despite their common fold and the conservation of active site residues. The majority of residues involved in substrate binding are conserved in asymmetrical Ap(4)A hydrolases from pathogenic bacteria, but are absent in their human counterparts, suggesting that it might be possible to generate compounds that target bacterial, but not human, Ap(4)A hydrolases.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

Catalysis by human leukocyte elastase: proton inventory as a mechanistic probe

Proton inventories (rate measurements in mixtures of H2O and D2O) were determined for the human leukocyte elastase catalyzed hydrolyses of thiobenzyl esters and p-nitroanilides of the peptides MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The dependencies of k2/Ks on mole fraction of solvent deuterium for the p-nitroanilides are "dome-shaped" and were fit to a model that incorporates the mechanistic features of generalized solvent reorganization when substrate binds to enzyme and partial rate limitation of k2/Ks by physical and chemical steps [Stein, R. L. (1985) J. Am. Chem. Soc. 107, 7768-7769]. The proton inventories for the deacylation of MeOSuc-Val-HLE and MeOSuc-Pro-Val-HLE are linear while those for the deacylation of MeOSuc-Ala-Pro-Val-HLE and MeOSuc-Ala-Ala-Pro-Val-HLE are "bowl-shaped" and could be fit to a quadratic dependence of rate on mole fraction of deuterium. These results are interpreted to suggest that the correct operation of the catalytic triad is dependent on substrate structure. Minimal substrates, which cannot interact with elastase at remote subsites, are hydrolyzed via a mechanism involving simple general-base catalysis by the active site histidine and transfer of a single proton in the rate-limiting transition state. In contrast, tri- and tetrapeptide substrates, which are able to interact at remote subsites, are hydrolyzed by a more complex mechanism of protolytic catalysis involving full functioning of the catalytic triad and transfer of two protons in the rate-limiting transition state. Finally, the proton inventories for the deacylation of MeOSuc-Ala-Pro-Ala-HLE and MeOSuc-Ala-Ala-Pro-Ala-HLE are dome-shaped and suggest that the chemical events of acyl-enzyme hydrolysis are only partially rate limiting for these reactions and that some other physical step is also partially rate limiting.