Half-site reactivity and the "induced-fit" hypothesis

Many subunit enzymes show the phenomenon of half-site reactivity, that is, the reaction with a substrate or substrate analogue shows a stoichiometry equal to one-half the number of “identical” subunits. One obvious potential explanation for this phenomenon is that reaction with one substrate molecule induces a change in an adjacent subunit, preventing a subsequent substrate molecule from reacting. Such an explanation has been proposed for the half-site reaction of the enzyme cytidine triphosphate synthetase with a substrate analogue (Levitzki et al., 1971). Analogous studies with glyceraldehyde-3-phosphate dehydrogenase reveal that the four active-site (Cys-149) sulfhydryl groups per tetrameric molecule react equivalently with iodoacetate whereas only two of the four sites undergo facile acylation with the substrate. The simple fact that the 2:1 stoichiometry ratio for the alkylation-acylation reactions is independent of the degree of prior alkylation rules out the ligand-induced asymmetry model as an explanation of the stoichiometries. Rather, it suggests that in muscle dehydrogenase there is a pre-existent non-equivalence among the subunits. On these bases we propose a procedure for distinguishing induced from pre-existent asymmetry in quaternary structure.     

Identification of mechanism of enzyme/transport process in the system of "enzyme/ transporter--substrate--effector" using the "ratio" method

Using equilibrium assumption the analysis of kinetics of enzyme/transport process has been developed. In the course of this process the enzyme can interact simultaneously with substrate S and effector E and as a result two products are generated: P1 (from substrate) and P2 (from effector) after catalytic effect of enzyme or transportation across the biological membrane. It has been demonstrated that the ratio (R = V0,1/V0,2) of initial rates of formation of reaction products P, (V0,1) and P2 (V0,2) represented in linearized form as a dependence on concentration of both substrate S0 and effector E0 allows to identify a specific mechanism of the present process. An algorithm of such identification has been developed.     

Mechanism of "uncoupled" tetrahydropterin oxidation by phenylalanine hydroxylase

Phenylalanine hydroxylase can catalyze the oxidation of its tetrahydropterin cofactor without concomitant substrate hydroxylation. We now report that this "uncoupled" tetrahydropterin oxidation is mechanistically distinct from normal enzyme turnover. Tetrahydropterins are oxygenated to 4a-carbinolamines only during catalytic events involving substrate hydroxylation. In the absence of hydroxylation tetrahydropterins are oxidized directly to quinonoid dihydropterins. Stoichiometry studies define a ratio of two tetrahydropterins oxidized per O2 consumed in uncoupled enzyme turnover, thus indicating the complete reduction of O2 to H2O. Complementary results establish the lack of H2O2 production by the enzyme when uncoupled and define a tetrahydropterin oxidase activity for the enzyme. Thus, the hydroxylating intermediate of phenylalanine hydroxylase may be discharged in two ways, by substrate hydroxylation or by electron abstraction. A mechanism is proposed for the uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase, and the significance of these findings is discussed.     

Assessment of protein-tyrosine phosphatase 1B substrate specificity using "inverse alanine scanning"

An "inverse alanine scanning" peptide library approach has been developed to assess the substrate specificity of protein-tyrosine phosphatases (PTPases). In this method each Ala moiety in the parent peptide, Ac-AAAApYAAAA-NH(2), is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 153 well defined peptides. The relatively small number of peptides allows the acquisition of explicit kinetic data for all library members, thereby furnishing information about the contribution of individual amino acids with respect to substrate properties. The approach was applied to protein-tyrosine phosphatase 1B (PTP1B) as a first example, and the highly potent peptide substrate Ac-ELEFpYMDYE-NH(2) (k(cat)/K(m) 2.2 +/- 0.05 x 10(7) M(-1) s(-1)) has been identified. More importantly, several heretofore unknown features of the substrate specificity of PTP1B were revealed. This includes the ability of PTP1B to accommodate acidic, aromatic, and hydrophobic residues at the -1 position, a strong nonpreference for Lys and Arg residues in any position, and the first evidence that residues well beyond the +1 position contribute to substrate efficacy.     

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.     

Molecular dynamics simulations of "loop closing" in the enzyme triose phosphate isomerase

We present molecular dynamics simulations on the active site region of dimeric triose phosphate isomerase (TIM) using the co-ordinates of native chicken muscle TIM as a starting point and performing simulations with no substrate, with dihydroxyacetone phosphate (DHAP), the natural substrate, and with dihydroxyacetone sulfate (DHAS), a substrate analog. Whereas most of the protein moves less than 1 A during the simulation, some residues in the active site loop move more than 8 A during the 10.5 picoseconds of dynamics for each of the simulations. Most interestingly, the nature of the loop motion depends on the substrate, with the largest motion found in the presence of DHAP, and only in the presence of DHAP does the loop move to "close off" the active site pocket. The final structure found for the DHAP-chicken TIM complex is qualitatively similar to that described by Alber et al. for DHAP-yeast TIM. Simulations on the monomeric protein gives insight into why the molecule is active only as a dimer.     

Chaperonin GroEL meets the substrate protein as a "load" of the rings

Chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. With the aid of cochaperonin GroES and ATP, double ring-shaped GroEL encapsulates non-native substrate proteins inside the cavity of the GroEL-ES complex. Although extensive studies have revealed the outline of GroEL mechanism over the past decade, central questions remain: What are the in vivo substrate proteins? How does GroEL encapsulate the substrates inside the cavity in spite of an apparent entropic difficulty? Is the folding inside the GroEL-ES cavity the same as bulk spontaneous folding? In this review I summarize the recent progress on in vivo and in vitro aspects of GroEL. In particular, emerging evidence shows that the substrate protein itself influences the chaperonin GroEL structure and reaction cycle. Finally I propose the mechanistic similarity between GroEL and kinesin, a molecular motor that moves along a microtubule in an ATP-dependent manner.     

A "cephalosporin-like" cyclic depsipeptide: synthesis and reaction with beta-lactam-recognizing enzymes

The cyclic depsipeptide 8-carboxy-3-phenylacetamido-3,4-dihydro-2H-1-benzopyran-2-one, a cyclic analog of aryl phenaceturates with structural similarity to cephalosporins, has been synthesized as a potential substrate/inhibitor of B-lactam-recognizing enzymes. It was found to be a tight-binding, poor substrate of class A beta-lactamases and an irreversible inhibitor of several DD-peptidases.     

"Survival" of mitochondria in vitro: physical and energy parameters

Isolated mitochondria have been maintained active and coupled for 72 hours at 25 C. Survival (retention of respiratory control) is a function of incubation temperature and dependent upon aeration and substrate. ATP does not entirely substitute for substrate, indicating a need for products of active metabolism other than energy. An improvement in respiratory control is often observed during the first several hours of incubation. Sedimentation and resuspension at 24-hour intervals prolonged survival. As revealed by electron microscopy, mitochondria maintained their basic structure during a 72-hour period at 25 C.     

Continuous production of ethanol by yeast "immobilized" in a membrane-contained fermentor

A dialysate-feed, immobilized-cell dialysis continuous fermentation system was investigated as a method of relieving product inhibition in the conversion of glucose to ethanol by cells of Saccharomyces cerevisiae ATCC 4126. The substrate was fed into a continuous dialysate circuit and then into a batch fermentor circuit via diffusion through the microporous membranes of an intermediate dialyzer. Simultaneously, product was withdrawn from the fermentor circuit through the dialyzer membranes into the dialysate circuit and out in the effluent. Since the fermentor was operated without an effluent, the cells essentially were immobilized and converted substrate to product by maintenance metabolism. Contrary to prior results with this novel system for the continuous fermentation of lactose to lactate by lactobacillus cells, a steady state of yeast cells in the fermentor did not occur initially but was obtained by the depletion of medium nitrogen and the prevention of cell breakage, although the substrate and product concentrations then became unsteady. The inherent advantages of the system was offset in the ethanol fermentation by relatively low productivity, which appeared to be limited by membrane permeability.     

Quantum chemical study of the "catalytic triad" of serine proteases

Using the semi-empirical MNDO/H method several systems simulating the reaction of tetrahedral intermediate formation in the active site of serine proteases have been studied. The role played by elements of the "catalytic triad" in increasing the reactivity of serine hydroxyl has been discussed. The formation of a strong hydrogen bond between His and Asp was shown to be important in lowering the activation energy in the reaction of Ser with substrate. The change in position of the proton located between Ser and His and between His and Asp was analysed. The influence of substrate distortion on the energy of intermediate formation has been considered.     

Schistosoma mansoni: proteinase activity of "hemoglobinase" from the digestive tract of adult worms

A method of collecting samples from the Schistosoma mansoni digestive tract was used to study proteinase activity. Activity against hemoglobin and a low molecular weight synthetic substrate, carbobenzoxy-arginyl-arginyl-7-amino-4-trifluoromethylcoumarin, was demonstrated in the soluble fraction of material regurgitated by S. mansoni adults and was dependent on the addition of a thiol compound, cysteine, to the assays. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography (AcA54), two proteins with estimated mol wt of 32,500 and 28,500 were found in the regurgitant and were associated with proteinase activity against both hemoglobin and the synthetic substrate. Homogenates of intact worms showed greater specific activity (synthetic substrate) in the females. Further, in bisected worms proteinase activity paralleled protein content, suggesting that, once secreted into the lumen, proteinase activity was distributed throughout the worm digestive tract.     

Computer modeling of the rhamnogalacturonase-"hairy" pectin complex

The structure of a pectin-bound complex of rhamnogalacturonase was modeled to identify the amino acid residues involved in catalysis and substrate binding. The "hairy" region of pectin, represented by six repeating stretches of (1-->4)-D-galacturonate-(1-->2)-L-rhamnose dimer, was flexibly docked into the putative binding site of rhamnogalacturonase from Aspergillus aculeatus whose X-ray structure is known. A search of the complex configurational space was performed using AutoDock for the dimeric and tetrameric sugar units in which the -1 galacturonate residue has various ring conformations. Then the plausible AutoDock solutions were manually extended to the dodecameric pectin models. Subsequently, the resulting complex models were subjected to solvated molecular dynamics using AMBER. In the best model, the substrate has an extended pseudo-threefold helix with the -1 ring in a 4H3 half-chair that approaches the transition state conformation. The catalytic machinery is clearly defined: Asp197 is a general acid and the activated water bound between Asp177 and Glu198 is a nucleophile. The active site is similar, with a small yet significant difference, to that of polygalacturonase that degrades the pectic "smooth" region of linear homopolymer of D-(1-->4)-linked galacturonic acid. Rhamnogalacturonase has ten binding subsites ranging from -3 to +7, while polygalacturonase has eight subsites from -5 to +3. The model suggests that the eight amino acids including three arginine and three lysine residues, all of which are invariantly conserved in the rhamnogalacturonase family of proteins, are important in substrate binding. The present study may aid in designing mutational studies to characterize rhamnogalacturonase.     

How to get all "A"s in polyadenylation

In an elegant study in this issue of Structure, Balbo and Bohm (2007) report the crystal structure of yeast poly(A) polymerase in a ternary complex with its substrate MgATP and the elongating poly(A) tail, providing molecular insights into the mechanism of polyadenylation.     

Characterization of covalent Ene adduct intermediates in "hydride equivalent" transfers in a dihydropyridine model for NADH reduction reactions

A study of the reactions of an NADH model, 1,4-di(trimethylsilyl)-1,4-dihydropyridine, 7, with a series of α,β-unsaturated cyano and carbonyl compounds has produced the first direct evidence for an obligatory covalent adduct between a dihydropyridine and substrate in a reduction reaction. The reactions were monitored by NMR spectroscopy. In all reactions studied, the covalent adduct was the first new species detected and its decomposition to form products could be observed. Concentrations of adducts were sufficiently high at steady-state that their structures could be determined directly from NMR spectra of the reaction mixtures; adduct structures are those expected from an Ene reaction between 7 and the substrate. This first reaction step results in transfer of the C(4) hydrogen nucleus of 7 to the substrate and formation of a covalent bond between C(2) of the dihydropyridine ring and the substrate α-atom. Discovery of these Ene-adduct intermediates completes the spectrum of mechanisms observed in NADH model reactions to span those with free radical intermediates, no detectable intermediates and now covalent intermediates. The geometry of the transition state for formation of the Ene adduct is compared with those of theoretical transition state models and crystal structures of enzyme-substrate/inhibitor complexes to suggest a relative orientation for the dihydropyridine ring and the substrate in an initial cyclic transition state that is flexible enough to accommodate all observed mechanistic outcomes.     

Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an "intersecting" N-acetylglucosamine residue to high mannose oligosaccharides

Glycoproteins synthesized by the cellular slime mold Dictyostelium discoideum have been shown to contain asparagine-linked high-mannose oligosaccharides which have an N-acetylglucosamine group in a novel intersecting position (attached beta 1-4 to the mannose linked alpha 1-6 to the core mannose). We have used crude membrane preparations from vegetative D. discoideum (strain M4) to characterize the enzyme activity responsible for catalyzing the transfer of GlcNAc to the intersecting position of high-mannose oligosaccharides. UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase activity in these preparations attaches GlcNAc to the mannose residue-linked alpha 1-6 to the beta-linked core mannose of the following Man9GlcNAc oligosaccharide as shown by the arrow. (formula; see text) It will also attach GlcNAc to the same intersecting position and/or to the bisecting position (beta-linked core mannose) of the following Man5GlcNAc oligosaccharide. (formula; see text) An analysis of the pH profiles, effects of heat denaturation, and substrate inhibitions on the addition of GlcNAc to either the intersecting or bisecting position of this Man5GlcNAc oligosaccharide indicates that a single enzyme activity is responsible for transferring GlcNAc to both positions. Various oligosaccharides were assayed to determine the substrate specificity of the transferase activity. These data indicate that both the mannose-attached alpha 1-3 and the mannose-attached alpha 1-6 to the mannose receiving the GlcNAc play a critical role in substrate suitability; absence of the alpha 1-6 mannose results in at least a 90% decrease in activity, while absence of the alpha 1-3 mannose results in a completely inactive substrate. This suggests that the minimal substrate is the disaccharide Man alpha 1-3Man.     

The "open" and "closed" structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii.

Recently, a new class of soluble inorganic pyrophosphatase (type-C PPase) has been described that is not homologous in amino acid sequence or kinetic properties to the well-studied PPases (types A and B) found in many organisms from bacteria to humans and thought to be essential to the cell. Structural studies of the type-C PPases from Streptococcus gordonii and Bacillus subtilis reveal a homodimeric structure, with each polypeptide folding into two domains joined by a flexible hinge. The active site, formed at the interface between the N and C-terminal domains, binds two manganese ions approximately 3.6 A apart in a conformation resembling binuclear metal centres found in other hydrolytic enzymes. An activated water molecule bridging the two metal ions is likely poised for nucleophilic attack of the substrate. Importantly, the S. gordonii and B. subtilis enzymes have crystallised in strikingly different conformations. In both subunits of the S. gordonii crystal structure (1.5 A resolution) the C-terminal domain is positioned such that the active site is occluded, with a sulphate ion bound in the active site. In contrast, in the B. subtilis structure (3.0 A resolution) the C-terminal domain is rotated by about 90 degrees, leaving the active site wide open and accessible for substrate binding.     

Kinetic manifestations of allosteric interactions in models of regulatory enzymes with "indirect" co-operativity

The shape of the plots of initial reaction rate (ν) versus initial substrate concentration ([S]₀) and versus initial concentration of allosteric effector ([F]₀) for the model of allosteric enzyme of Monod, Wyman &; Changeux (1965) and for the model of dissociating regulatory enzyme has been analysed by means of the inconstant exponent (q) for substrate or effector concentration, respectively. It has been shown that allosteric interactions in above-mentioned models with “indirect” co-operativity may be manifested not only by the sigmoidal shape of the plot of ν versus [S]₀ or ν versus [F]₀ (with one point of inflexion) but also by the increase in the magnitude of exponent q in progress of saturation process of the enzyme by the substrate or by the effector in the absence of the sigmoidal shape of these plots. It has been shown also that the plot of ν versus [S]₀ has two inflexion points when the parameters have certain definite values. One of these inflexion points (or even both at definite values of the parameters) is hardly discernible. At certain definite values of the parameters two inflexion points may be kinetically manifested by such phenomenon as “negative” co-operativity (q < 1). This is possible if one of the interconvertable enzyme forms exceeds another not only in the affinity to the substrate but also in the value of the rate constant for catalytic breakdown of the enzyme-substrate complex.     

Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation

The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.     

Renin substrate (angiotensinogen) preparations in the determination of prorenin and renin: evidence for extrarenal plasma prorenin and its renal "convertase"

Standard methods for determining prorenin-renin concentrations in plasma (PRC) and other tissues require the addition of exogenous renin substrate (angiotensinogen) to improve the kinetics of the renin reaction. We studied the effects of substrate prepared from normal human plasma fraction Cohn IV-4, or from nephrectomized (2NX) sheep plasma, on PRC of normal and 2NX human plasmas before and after prorenin activation by acid, cold, and trypsin, and compared the results with plasma renin activities (PRA, no added substrate). Plasmas from 2NX men exhibited negligible basal PRA, indicating that very little, if any, renin had been formed from the extrarenal prorenin they contained, and suggesting the lack of an endogenous prorenin activating mechanism, or "convertase," of probable renal origin. Prorenin was demonstrable by tryptic activation, more than by acid or cold, at up to about 30% of normal. Addition of Cohn IV-4 substrate to 2NX plasma unexpectedly produced (i) a basal PRC value higher than in normal plasma, (ii) total renin values after activation by acid, cold, and trypsin that were much closer to normal values than reflected by PRA methodology, without a commensurate increase (if anything a decrease) in prorenin as a percentage of total renin estimated by all activation methods, and (iii) substantial equalization of activation effects such that trypsin was no longer more effective than acid and cold (and this was also noted with normal plasma). The skewing effect of adding Cohn IV-4 substrate on the PRC of 2NX plasma was much greater than in normal plasma, even though 2NX plasma already had an above normal level of endogenous substrate and should have been influenced less. Enhancement of PRC was very pronounced even when Cohn IV-4 was added to make up only 9% of total (endogenous + exogenous) substrate in the incubation system, suggesting that it was not the added substrate but a renin-generating contaminant that inflated the PRC. Such inflation could be blocked by adding protease inhibitors, suggesting that the responsible protease(s) acted as a prorenin "convertase" that generated new renin from renal and (or) extrarenal prorenin contributed by the added substrate, as well as by the plasma being assayed. One component of convertase could be kallikrein, which was identified by chromogenic assay, the importance of which relative to total convertase activity is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)