The seed germination model of enzyme catalysis.

The activity of enzymes and other biological macromolecules is often sensitively dependent on physiochemical context. Seed germination provides an analogy that helps to elicit the control and information processing capabilities of enzymatic networks. Like a seed, the enzyme takes a particular action (complexes with a specific substrate and catalyzes a specific reaction) when a specific set of milieu influences is satisfied. The context sensitivity, specificity and speed are enormously enhanced by the parallelism inherent in the electronic wave function (i.e. by the superposition of electronic states). This parallelism is converted to speedup through electronic-conformational interactions. The quantum speedup effect allows biological 'switches' to have qualitatively greater pattern recognition capabilities than electronic switches. Consequently the information processing and control capabilities of biomolecular systems exceed the capabilities obtainable from classical models and exceed the intuitive expectations that have developed through the study of such models.     

A linearization method for low catalytic activity enzyme kinetic analysis

A kinetic analysis was made and a linear plot based on the general rate equation derived by Laidler [Can. J. Chem. 33, 1614-1624] is proposed. This linearization method allows determining the kinetic parameters (K(m), k(cat)) and [E](0) for enzymes with low catalytic activity. The method was applied to chloroperoxidase from Caldariomyces fumago [EC 1.11.1.10], whose kinetic parameters K(m)(app), k(cat)(app), and [E](0) with monochlorodimedone as substrate, were obtained by using the linearization plot and the V(max) value (calculated by Eadie-Hofstee plot). This plot could also be useful to the study of abenzyme kinetics provided the concentration of the latter is either higher or equal than K(m) value.     

pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.     

Microenvironmental effects on enzyme catalysis. A kinetic study of hydrophobic derivatives of chymotrypsin

The aim of this work was to study the role of hydrophobic interactions in the enzymic activity of chymotrypsin. The amino groups of chymotrypsin were chemically modified by aliphatic aldehydes of various chain lengths - acetaldehyde, butyraldehyde, hexanal - and with two aldehydes of different steric hindrance - benzaldehyde and trimethyl acetaldehyde. After a rapid study of the derivated enzymes, the hexylchymotrypsin has been chosen for its new catalytic properties: the Michaelis constant is not modified and the maximal velocity with N-glutaryl-L-phenylalanine-4-nitroaniline is increased to 164%. The increase is due to the increase of the acylation constant, k2, by 230%. The value of k3 is not modified or less modified. In the modified enzyme, 85% of free amino acids are still able to react with trinitrobenzenesulphonic acid. The optimum pH is shifted by one pH unit towards the alkaline pH. The thermodynamic study shows that the catalytic process itself is not modified. The increase in Vm could be a simple increase of k2 linked to a modification of the site or of the protein. The phenomenon described is very specific and obtained only with one modification, hexanal, and with one enzyme, alpha-chymotrypsin.     

A microcomputer method for designing optimal experiments for estimating enzyme kinetic parameters

A computer program, written in BASIC, for designing optimal experiments with the aim of evaluating estimates of the parameters for any enzyme kinetic model is given. This computer program can be run on any microcomputer with less than 32 Kbytes of random access memory. The program uses the termed D-optimization design criterion, which minimizes the determinant of the variance-covariance matrix. The user only supplies the rate equation, the maximum and minimum concentrations of substrates and inhibitors, the weighting pattern, and the best possible values of the parameters. The computer supplies the optimal substrate and inhibitor concentrations (one for each parameters), for estimating the parameter values, and the determinant of the variance-covariance matrix. Likewise, the microcomputer supplies the eigenvalues and eigenvectors of information and redundancy matrices, the sensitivity and the global redundancy.     

An enzyme kinetic model for describing fermentation processes

An enzyme kinetic model has been proposed to describe fermentation processes. This type of model was chosen because it is biologically sound, can incorporate all of the important engineering control variables, and can draw upon, in its development, the extensive kinetic literature. An intial qualitative test for this model was made on the gluconic acid fermentation. A necessary check of the model was that Monod's empirical cell growth and yield equations were derived as a special case. The model also offered an explanation for the hysteresis behavior of the gluconic acid production rate as a function of gluconolactone.     

The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability

Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of alpha-1,6-branch points. They are the members of the alpha-amylase family and have four conserved regions in a central (beta/alpha)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (DeltaN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same K(m) values as PvSBE2 but had lower V(max) values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.     

Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme.

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) Biochemistry 35, 4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 17O-labeled forms of phosphoenolpyruvate show that Mn(2+), bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of 2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of 2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn(2+) gives lower maximal activity than Mg(2+), apparently due to slow release of Zn(2+) from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn(2+) from the enzyme.     

The role of metal in liver alcohol dehydrogenase catalysis. Spectral and kinetic studies with cobalt-substituted enzyme.

The kinetic and spectral properties of native and totally cobalt-substituted liver alcohol dehydrogenase have been compared. Based on titrimetric determinations of enzyme active site concentration, the turnover number at pH 7.0 for cobalt enzyme was the same as for native enzyme. At pH 10, however, the turnover number was slower for cobalt-substituted enzyme, 3.14 s-1 as compared with 4.05 s-1 for native enzyme. A comparison between native and totally cobalt-substituted enzyme showed a blue-shifted enzyme-NADH double difference spectrum and a splitting and red-shifted enzyme-NAD+-pyrazole double difference spectrum in the near-ultraviolet. The 655-nm peak of the cobalt-substituted enzyme was perturbed by the formation of enzyme-NADH binary complex, enzyme-NAD+-trifloroethanol ternary complex, and enzyme-NAD+ binary complex formation. At pH 7.0, the only observable step in the reaction sequence with a significantly different rate constant for cobalt enzyme was the catalytic hydrogen-transferring step. The rate constant for this step is 92 s-1 for totally cobalt-substituted enzyme as compared with 138 s-1 for native liver alcohol dehydrogenase. The results of this study indicate that zinc is involved in catalysis alcohol and NADH.     

Unstable electron pairing and the energy loan model of enzyme catalysis

A theory of enzyme catalysis is described which utilizes a thermodynamically consistent construction of a free energy diagram with different pathways for complex formation and decomposition. The switch to the decomposition pathway occurs when downward uncertainty and thermal fluctuations make possible a short-lived potential energy dominance in which parallel spin electrons are paired and thus free to drop below the energy floor normally maintained by the Pauli exclusion principle. Such pairing is possible if van der Waal's and other weak interactions holding the complex together impose confinement constraints on parallel spin electrons, thereby both increasing uncertainty fluctuations in their kinetic energy and weakly favoring a phase correlation in their motion (which can be interpreted in terms of an exchange of virtual particles). The paired configuration is highly unstable and thus energy released by pair falling is either immediately recaptured to re-establish a normal orbital structure or, if the pair persists long enough to produce a nuclear motion, recaptured at the end of this motion. In the latter case the release of energy can be thought of as an energy loan which finances the switch to the lower activation energy pathway without compromising an energy-balanced regeneration of the enzyme. The advantage is that the complex (because of its instability) has a real free energy which is lower than the free energy which would be assigned to it on the basis of its equilibrium concentration. This increases the specificity and speed of complex formation without decreasing the speed of decomposition. The theory predicts that the magnetic moment which marks the pair should accompany the nuclear (e.g. allosteric) motion and that the pair formation stage of the enzymatic process should have an anomalous temperature dependence. Variations of the model may be constructed to deal with a number of processes involving macromolecular motions, including sequential processes in catalysis, allosteric control, persistent molecular motions, self-assembly, energy transfer, channeled transport, and protection against inhibitors.     

Hydrogen-bonding in enzyme catalysis. Fourier-transform infrared detection of ground-state electronic strain in acyl-chymotrypsins and analysis of the kinetic consequences.

I.r. difference spectra are presented for 3-(indol-3-yl)acryloyl-, cinnamoyl-, 3-(5-methylthien-2-yl)acryloyl-, dehydrocinnamoyl- and dihydrocinnamoyl-chymotrypsins at low pH, where the acyl-enzymes are catalytically inactive. At least two absorption bands are seen in each case in the ester carbonyl stretching region of the spectrum. Cinnamoyl-chymotrypsin substituted at the carbonyl carbon atom with 13C was prepared. A difference spectrum in which 13C-substituted acyl-enzyme was subtracted from [12C]acyl-enzyme shows two bands in the ester carbonyl region and thus confirms the assignment of the features to the single ester carbonyl group. The frequencies of the ester carbonyl bands are interpreted in terms of differential hydrogen-bonding. In each case a lower-frequency relatively narrow band is assigned to a productive potentially reactive binding mode in which the carbonyl oxygen atom is inserted in the oxyanion hole of the enzyme active centre. The higher-frequency band, which is broader, is assigned to a non-productive binding mode in each case, where a water molecule bridges from the carbonyl oxygen atom to His-57; this mode is equivalent to the crystallographically determined structure of 3-(indol-3-yl)acryloyl-chymotrypsin, i.e. the Henderson structure. A difference spectrum of dihydrocinnamoyl-chymotrypsin taken at higher pH shows resolution of a feature centred upon 1731 cm-1, which is assigned to a non-bonded conformer in which the carbonyl oxygen atom is not hydrogen-bonded. Perturbation of the protein spectrum in the presence of acyl groups is interpreted in terms of enhanced structural rigidity. It is reported that the ester carbonyl region of the difference spectrum of cinnamoyl-subtilisin is complicated by overlap of features that arise from protein perturbation. Measurements of carbonyl absorption frequencies in a number of solvents of the methyl esters of the acyl groups used to make acyl-enzymes have permitted determination of the apparent dielectric constants experienced by carbonyl groups in the enzyme active centre as well as a discussion of the effects of polarity. The ester carbonyl bond strengths of the various conformations were estimated by using simple harmonic oscillator theory and an empirical relation between the force constants and bond strengths. The fractional bond breaking induced by hydrogen-bonding was used to calculate rate enhancement factors by using absolute reaction rate theory.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.     

Neutron crystallography for the elucidation of enzyme catalysis

Hydrogen atoms and hydration water molecules in proteins are indispensable for many biochemical processes, especially enzymatic catalysis. The locations of hydrogen atoms in proteins are usually predicted based on X-ray structures, but it is still very difficult to know the ionization states of the catalytic residues, the hydration structure of the protein, and the characteristics of hydrogen-bonding interactions. Neutron crystallography allows the direct observation of hydrogen atoms that play crucial roles in molecular recognition and the catalytic reactions of enzymes. In this review, we present the current status of neutron crystallography in structural biology and recent neutron structural analyses of three enzymes: ascorbate peroxidase, the main protease of severe acute respiratory syndrome coronavirus 2, and copper-containing nitrite reductase.     

Higher metal-ligand coordination in the catalytic site of cobalt-substituted Thermoanaerobacter brockii alcohol dehydrogenase lowers the barrier for enzyme catalysis

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP(+)/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.     

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme,benzoylformate decarboxylase

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.