Intermediate in the O-O bond cleavage reaction of an extradiol dioxygenase

The reactive oxy intermediate of the catalytic cycle of extradiol aromatic ring-cleaving dioxygenases is formed by binding the catecholic substrate and O2 in adjacent ligand positions of the active site metal [usually Fe(II)]. This intermediate and the following Fe(II)-alkylperoxo intermediate resulting from oxygen attack on the substrate have been previously characterized in a crystal of homoprotocatechuate 2,3-dioxygenase (HPCD). Here a subsequent intermediate in which the O-O bond is broken to yield a gem diol species is structurally characterized. This new intermediate is stabilized in the crystal by using the alternative substrate, 4-sulfonylcatechol, and the Glu323Leu variant of HPCD, which alters the crystal packing.     

Influence of substrate binding on the mechanical stability of mouse dihydrofolate reductase

We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy. We find that under mechanical forces dihydrofolate reductase unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the measured length increase of approximately 22 nm we suggest a structure for this intermediate with intact substrate binding sites. In the presence of the substrate analog methotrexate and the cofactor NADPH lifetimes of this intermediate are increased by up to a factor of two. Comparing mechanical and thermodynamic stabilization effects of substrate binding suggests mechanical stability is dominated by local interactions within the protein structure. These experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.     

Sequential substrate utilization and effectiveness factor in fixed biofilms

A mathematical model describing the change in effectiveness factor or effective diffusivity for the intermediate substrate in sequential substrate removal reactions in fixed biofilms is developed. Typical results from the numerical solution of the resulting equation are presented. The production of intermediate substrate in the biofilm increases the conversion of primary substrate to ultimate product in all cases, but the increase is not always significant. Using typical data applying to laboratory anaerobic fermentation studies, it is demonstrated that a small but significant change in reaction kinetics and the effectiveness factor exists.     

Comparative studies on metabolic activity of planktonic,benthic and epiphytic bacteria

Summary Differences have been noted in metabolic activity of bacteria isolated from water, mud and from Canadian pondweed (Elodea canadensis).The epiphytic bacteria showed the greatest metabolic activity in every instance, benthic bacteria were least active and the water isolates were intermediate.Non-chromogenic bacteria were metabolically more active than the chromogens.Casamino acids proved to be the most readily oxidized substrate among the compounds studied. Glucose, succinate, fumarate and gluconate were readily used by most epiphytic and planktonic bacteria. Acetate was intermediate and fructose was the least suitable substrate for these organisms. Casamino acids proved to be the best substrate for all three groups, although the benthic bacteria were less active with this substrate than the former two groups of bacteria. Gluconate, succinate, pyruvate and acetate were intermediate, and glucose and fructose were least suitable for most of the benthic bacteria.     

Structural determinants of substrate binding to Bacillus cereus metallo-beta-lactamase

Binding and hydrolysis of the beta-lactams cefotaxime, cephapirin, imipenem, and benzylpenicillin by the metallo-beta-lactamase from Bacillus cereus were studied by presteady state kinetic measurements. In all cases, the substrate was unmodified in the most populated reaction intermediate, and no chemically modified substrate species accumulated to a detectable amount. The cephalosporins tested showed similar formation rate constants for this intermediate, and they differed mostly in their decay rates. Formation of a non-productive enzyme.substrate complex was detected for imipenem. The substrate binding differences can be accounted for by considering the structural features of each substrate. The apoenzyme could not bind any of the substrates, but binding was restored when the apoenzyme was reconstituted with Zn(II), revealing that the metal ions are the main determinants of substrate binding. This evidence is in line with the lack of an optimized substrate recognition patch in B1 and B3 metallo-beta-lactamases that provides a broad substrate spectrum.     

Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization

Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.     

A kinetic study of hypoxanthine oxidation by milk xanthine oxidase.

The course of the reaction sequence hypoxanthine----xanthine----uric acid catalysed by xanthine:oxygen oxidoreductase from milk was investigated on the basis of u.v. spectra taken during the course of hypoxanthine and xanthine oxidations. It was found that xanthine accumulated in the reaction mixture when hypoxanthine was used as a substrate. The time course of the concentrations of hypoxanthine, xanthine intermediate and uric acid product was simulated numerically. The mathematical model takes into account the competition of substrate, intermediate and product and the accumulation of the intermediate at the enzyme. This type of analysis permits the kinetic parameters of the enzyme for hypoxanthine and xanthine to be obtained.     

Structural and mechanistic studies of enolase

The high-resolutionstructure of yeast enolase cocrystallized with its equilibrium mixture of substrate and product reveals the stereochemistry of substrate/product binding and therefore the groups responsible for acid/base catalysis and stabilization of the enolate intermediate. Expression and characterization of site-specific mutant forms of the enzyme have confirmed the roles of amino acid side chains in the catalysis of the first and second steps of the reaction. Coordination of both required magnesium ions to the carboxylate of the substrate/product indicates a role for these cations in stabilization of the intermediate.     

Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.     

Inactivation of pig heart alanine aminotransferase by beta-chloroalanine.

The substrate analog β-chloro-l-alanine rapidly inactivates the pig heart alanine aminotransferase. Inactivation is dependent upon formation of an intermediate. The substrate alanine protects the enzyme by competing with the inhibitor for the formation of this intermediate. Halide and carboxylate anions accelerated the rate of inactivation once the enzyme-inhibitor complex was formed. This acceleration appears to mimic the action with the substrate, for the rate of exchange transamination between unlabeled alanine and labeled pyruvate is similarly accelerated. When labeled inhibitor was used, the inactive enzyme became labeled. The spectral changes which occur resemble, in many respects, those which occur with aspartate aminotransferase when its active-site lysine undergoes alkylation by β-chloroalanine. We conclude that chloroalanine fulfills the criteria for a “suicide substrate.”     

Diethyl-p-nitrophenyl phosphate: an active site titrant for lipoprotein lipase

Diethyl-p-nitrophenyl phosphate is an active site-directed irreversible inhibitor of bovine milk lipoprotein lipase catalyzed hydrolysis of the water-soluble substrate, p-nitrophenyl butyrate. Interaction of lipoprotein lipase and the inhibitor in the absence of substrate gives a biphasic kinetics profile, which is consistent with rapid formation of a phosphoryl-lipoprotein lipase intermediate which hydrolyzes slowly. The magnitude of the absorbance increase accompanying formation of the intermediate provides an analytical method for determining lipoprotein lipase active site concentration.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.     

Kinetic competence of enzymic intermediates: fact or fiction?

A number of enzymatic reactions involve intermediates that are not normally released during the reaction. Whether such an intermediate when added to the enzyme reacts as fast or faster than the normal substrates, and thus is "kinetically competent", depends on the degree to which the equilibrium constant for forming the intermediate from the substrates is different on the enzyme surface and in solution, as well as on the relative affinities of the enzyme for substrate and intermediate. Similar values for these equilibrium constants require that the intermediate react slowly, while a far more favorable value for intermediate formation on the enzyme allows the intermediate to react at up to the diffusion-limiting rate. When one intermediate is formed from two substrates, it may react much more rapidly than when two intermediates are formed from two substrates, or one from one. Comparison of the kinetics of the putative intermediate(s) and the substrate(s) can reveal a great deal about the mechanism of the catalytic reaction and the kinetic barrier that normally keeps the intermediate(s) on the enzyme.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

Introducing total substrates simplifies theoretical analysis at non-negligible enzyme concentrations: pseudo first-order kinetics and the loss of zero-order ultrasensitivity

The Briggs–Haldane standard quasi-steady state approximation and the resulting rate expressions for enzyme driven biochemical reactions provide crucial theoretical insight compared to the full set of equations describing the reactions, mainly because it reduces the number of variables and equations. When the enzyme is in excess of the substrate, a significant amount of substrate can be bound in intermediate complexes, so-called substrate sequestration. The standard quasi-steady state approximation is known to fail under such conditions, a main reason being that it neglects these intermediate complexes. Introducing total substrates, i.e., the sums of substrates and intermediate complexes, provides a similar reduction of the number of variables to consider but without neglecting the contribution from intermediate complexes. The present theoretical study illustrates the usefulness of such simplifications for the understanding of biochemical reaction schemes. We show how introducing the total substrates allows a simple analytical treatment of the relevance of significant enzyme concentrations for pseudo first-order kinetics and reconciles two proposed criteria for the validity of the pseudo first-order approximation. In addition, we show how the loss of zero-order ultrasensitivity in covalent modification cycles can be analyzed, in particular that approaches such as metabolic control analysis are immediately applicable to scenarios described by the total substrates with enzyme concentrations higher than or comparable to the substrate concentrations. A simple criterion which excludes the possibility of zero-order ultrasensitivity is presented.     

Kinetic evidence for an acyl-enzyme intermediate in D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus.

The kinetics of hydrolysis and transpeptidation of the synthetic substrate diacetyl-L-lysyl-D-alanyl-D-alanine and of the natural substrate UDP-acetylmuramyl pentapeptide and related compounds catalyzed by the D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus in the presence of the nucleophiles hydroxylamine or glycine have been examined. These kinetic data suggest that an acyl-enzyme intermediate is formed in the first step of the reaction and that the transpeptidation is the consequence of the partitioning of this intermediate between water and the nucleophile in the second step.     

Inhibitors can activate proteases to catalyze the synthesis and hydrolysis of peptides

Competitive inhibitors can activate proteases (papain, trypsin, and cathepsin S) to catalyze the synthesis of peptide bonds and accelerate the hydrolysis of poor substrates (from 1 to 99%). Reaction mixtures contained intermediate molecules that were formed by the coupling of the inhibitor with the poor substrate. This and other findings suggest the following chain of events. Part of the binding energy of formation of the enzyme-inhibitor complex was used to activate the inhibitor, i.e., to form acyl-enzyme species with a high-energy bond (e.g., a thioester bond in the case of papain) required for coupling the inhibitor with the substrate to form the intermediate molecule. The latter was subjected to successive reactions which led to a stepwise degradation of the substrate, as well as to the regeneration of the inhibitor. One mole of the inhibitor could catalyze rapid hydrolysis of at least 53 mol of substrate. The intermediate molecules were the species undergoing rapid hydrolysis. Therefore, 1 mol of inhibitor was involved in the synthesis of 53 mol of intermediate molecules; i.e., the inhibitor functioned as a cofactor that catalyzed the synthesis of peptides. Thus, the binding energy of formation of the enzyme-inhibitor complex can be utilized to catalyze the synthesis of peptide bonds in the absence of an exogenous energy source (e.g., ATP).     

Morpholinecarbonyl-Rhodamine 110 based substrates for the determination of protease activity with accurate kinetic parameters

Commonly used fluorogenic substrate analogues for the detection of protease activity contain two enzyme-cleavable bonds conjugated to the fluorophore. Enzymatic cleavage follows a two-step reaction with a monoamide intermediate. This intermediate shows fluorescence at the same wavelength as the final product complicating the kinetic analysis of fluorescence-based assays. Fluorogenic substrate analogues for α-chymotrypsin with one cleavable peptide bond have been prepared from morpholinecarbonyl-Rhodamine 110 (MC-Rh110). A comparison of their kinetic properties with the corresponding (peptide)(2)-Rh110 derivatives revealed that these frequently used double-substituted substrate analogues yield only apparent K(m) and k(cat) values that are quite different from the kinetic parameters obtained from the monosubstituted MC-Rh110 based substrate analogues. Although both the monoamide intermediate and MC-Rh110 are monosubstituted Rhodamine 110 derivatives, they show different spectroscopic properties. The data from the spectroscopic analysis clearly show that these properties are directly related to the electron structure of the fluorophore and not to the previously proposed equilibrium between the lactone form and the open ionic form of the fluorophore. This knowledge about the determinants of the spectroscopic properties of monosubstituted Rhodamine 110 introduces a way for a more systematic development of new fluorogenic protease substrate analogues.     

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.     

Phosphorylated threonine as the covalent intermediate in snake venom phosphodiesterase

The covalent intermediate of snake venom phosphodiesterase has been isolated using thymidine 5'-[alpha-32P]triphosphate as substrate. Phosphoamino acid analysis of the labeled enzyme demonstrates that threonine is the active site residue forming the covalent intermediate. 5'-Nucleotide phosphodiesterase is the first enzyme reported to have an active site threonine forming a covalent intermediate.