Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.     

Steady state and pre-steady state kinetic properties of rat liver selenium-glutathione peroxidase.

The kinetic properties of partially purified rat liver selenium-glutathione peroxidase were studied under various conditions. Steady state kinetic measurements show sigmoidal saturation curves, parabolic double reciprocal plots, and Hill coefficients greater than unity. Although these kinetic results appear to show cooperative interactions between subunits, they more reflect the presence of several oxidation-reduction forms of the catalytic site. A substrate-induced transition between enzyme forms was evidence by the occurrence of a lag in the attainment of the final steady state velocity under certain preincubation conditions. This hysteretic behavior was evident only when the enzyme was incubated in the absence of reduced glutathione, the donor substrate. Thus, reduced glutathione induces the transition to the fully active form of the enzyme, a slow process requiring about 0.5 min after addition of glutathione, depending on conditions. The length, tau, of the lag period is dependent on the concentrations of enzyme and glutathione, but to a first approximation, this lag period is independent of the concentration of the hydroperoxide acceptor substrate. The lag period is also relatively independent of the nature of the hydroperoxide species. A model for the transition process that is compatible with these observations and with the possible oxidation-reduction properties of the selenium moiety of the enzyme is suggested.     

Oxidation by mushroom tyrosinase of monophenols generating slightly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of mettyrosinase (Em) and oxytyrosinase (Eox) that exists in the native form of the enzyme. The latter form is active on monophenols although the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex as the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as 4-methoxyphenol, 4-ethoxyphenol and 4-tert-butylphenol, tyrosinase generates o-quinones which become unstable with small constants of approximately < 10-3 s-1. The system evolves from an initial steady state, reached when t-->0, through a transition state towards a final steady state, which is never reached because the substrate is largely consumed. The mechanisms proposed to explain the enzyme's action can be differentiated by the kinetics of the first steady state. The results suggest that tyrosinase hydroxylates monophenols to o-diphenols, generating an intermediate Em-diphenol in the process, which may oxidize the o-diphenol or release it directly into the medium. In the case of o-quinone formation, its slow instability generates o-diphenol which activates the enzymatic system yielding parabolic time recordings.     

Role of protein conformational dynamics in the catalysis by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

Enzymatic catalysis has conflicting structural requirements of the enzyme. In order for the enzyme to form a Michaelis complex, the enzyme must be in an open conformation so that the substrate can get into its active center. On the other hand, in order to maximize the stabilization of the transition state of the enzymatic reaction, the enzyme must be in a closed conformation to maximize its interactions with the transition state. The conflicting structural requirements can be resolved by a flexible active center that can sample both open and closed conformational states. For a bisubstrate enzyme, the Michaelis complex consists of two substrates in addition to the enzyme. The enzyme must remain flexible upon the binding of the first substrate so that the second substrate can get into the active center. The active center is fully assembled and stabilized only when both substrates bind to the enzyme. However, the side-chain positions of the catalytic residues in the Michaelis complex are still not optimally aligned for the stabilization of the transition state, which lasts only approximately 10(-13) s. The instantaneous and optimal alignment of catalytic groups for the transition state stabilization requires a dynamic enzyme, not an enzyme which undergoes a large scale of movements but an enzyme which permits at least a small scale of adjustment of catalytic group positions. This review will summarize the structure, catalytic mechanism, and dynamic properties of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and examine the role of protein conformational dynamics in the catalysis of a bisubstrate enzymatic reaction.     

The dimeric form of phosphoenolpyruvate carboxylase isolated from maize: physical and kinetic properties

Phosphoenolpyruvate carboxylase purified from maize was a homodimer of molecular weight 200 kDa and was readily converted to a tetrameric form in the presence of Mg2+ plus PEP or Mg2+ alone. During the assay, the enzyme activity increased with time, reaching a steady state after a discernible lag, suggesting its hysteretic nature. The hystereses was not due to oligomerization of the enzyme as the lag time tau was independent of the enzyme concentration and the lag was not abolished on preincubation with 25 mM Mg2+, the condition under which the enzyme existed in tetrameric form. Nevertheless, the lag could be abolished on preincubating the enzyme with PEP plus Mg2+, indicating that the hystereses is due to a PEP plus Mg2(+)-induced slow transition of the enzyme to an activated state during the catalysis. During steady state, the enzyme showed cooperative kinetics for PEP and Mg2+ at pH 7. It had two binding sites with nearly 10-fold difference in affinities for PEP and Mg2+.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).     

Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period.     

Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea: Further Evidence on the Dimer-Tetramer Interconversion

Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at −70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage—which is composed of an equilibrium mixture of dimer and tetramer—with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. Thus the question of whether some covalent modification of the enzyme, such as phosphorylation, is required to permit complete changes in equilibrium remains open.     

Transition state of the glycolytic pathway under FDP saturating conditions: experimental studies and a theoretical model

1. The transition state of the glycolytic pathway, under FDP saturating conditions, from no ADP to ADP-saturating levels, is studied in a metabolic model in vitro obtained from rat skeletal muscle. 2. When ADP is absent from the reaction mixture a steady state for NADH concentration is observed. After ADP addition, a new steady state is reached. The transition state from the first steady state to the second one shows a pulse of NADH. Both the profile and the size of this pulse depend on the enzyme concentration. 3. A kinetic model of the lower part of glycolysis (after PFK reaction) is proposed, and this is described by a set of first order coupled nonlinear differential equations. The results obtained through stability analysis and numerical integration of these equations agree with the experimental ones. 4. The possible role of the above mentioned transition state on the transmitter mechanism of glycolytic oscillations from PFK to the lower part of the glycolysis is discussed.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Hysteretic properties of NADP-malic enzyme from sugarcane leaves

NADP-malic enzyme highly purified from sugarcane leaves exhibited hysteretic properties. This behavior resulted in a lag phase during activity measurement of the enzyme preincubated in the absence of substrates. The lag was inversely proportional to the protein concentration during preincubation, which suggests that changes in the aggregational state of the enzyme are responsible for hysteresis. The pH conditions as well as the presence of different compounds in the preincubation medium modified the hysteretic properties of the enzyme. Mg²⁺ eliminated the lag period and increased the enzyme activity by nearly 2-fold. NADP⁺, 3-phosphoglycerate, ATP and dithiothreitol shortened the lag phase. The substrate l-malate inhibited the enzyme by decreasing the steady state velocity and increasing the lag time in a concentration-dependent manner. NADPH, triose-phosphates and high ionic strength increased the lag phase. Results are consistent with the view that the level of different metabolites and the pH conditions at the chloroplast regulate the activity of NADP-malic enzyme in a coordinate and effective manner.Abbreviations Diamide azodicarboxylic acid bis(dimethylamide) - DHAP dihydroxyacetone-phosphate - DTT dithiothreitol - Ga3P glyceraldehyde-3-phosphate - NADP-ME NADP-dependent malic enzyme - PEP phosphoenolpyruvate - 3PGA 3-phosphoglycerate     

Coenzyme-induced slow transitions of NADP-sorbitol dehydrogenase from Gluconobacter oxydans]

The kinetic properties of NADP-dependent sorbitol dehydrogenase from G. oxydans cell extract were studied at pH 8.8 and 9.3 in the direction of D-sorbitol oxydation. It was shown that the shape of the kinetic curves of NADPH accumulation in time is characterised by initial burst whose magnitude depends on the concentration of the enzyme extract used. Preincubation of the enzyme with NADP or D-sorbitol eliminated the initial burst on these curves and transformed them into straight lines coming from the start of co-ordinates. The dependence of the stationary reaction rate on the enzyme extract concentration is not a linear one. The kinetic dependences of stationary rate of the reaction catalysed by the enzyme on the concentration of D-sorbitol and NADP at pH 8.8 and 9.3 were examined under all conditions studied; the shape of these kinetic curves altered to considerable extent with the alteration of the enzyme extract concentration in the reaction mixture and pH. At pH 9.3 several intermiediate plateaux were found on the curves of the D-sorbitol concentration dependent stationary rate of the reaction. The preincubation of the enzyme extract with NADP during 1.5 h removed the intermediate plateau on these curves and made them hyperbolic. Disk-electrophoresis of the enzyme extract in PAAG concentration gradient showed that at pH 8.8 the enzyme exists in one active form, while at pH 9.3 it exists in three major and three minor active forms of the enzyme differing in their molecular weights are found. It is assumed that the enzyme from G. oxydans cell extract can exist in a great number of molecular equilibrium forms, the rate of quilibrium being comparable or significantly less than that of the enzymatic reaction. NADP significantly influences on the equilibrium of the molecular forms of the enzyme.     

Efficient lipase catalysed production of a lubricant and surfactant formulation using a continuous solvent-free process

The transesterification of sunflower oil with a high oleic acid residue content (typically 83.5%) with butanol-1 by immobilised Lipozyme was carried out in a solvent free system and in a continuous way. During the first 6 h of reactor operation, a transition phase was observed, in which the main products were butyl ester and glycerol. This latter being insoluble in the reaction mixture, it is adsorbed onto the enzyme support thus leading to a decrease in enzyme performance. Step by step, less and less glycerol is produced and finally when glycerol is no longer produced a steady state is attained. The product composition is a mixture of butyl ester (65 molar%), monoglyceride (26 molar%), diglyceride (6 molar%) and residual triglyceride (3 molar%). This mixture has interesting lubricant and surfactant properties. The reactor was maintained without any loss in activity for a period of 3 months. This result is very different to that obtained using an organic solvent (n-hexane) which leads to a total loss of enzyme activity within a few hours.     

Stopped flow and steady state kinetic studies of the effects of metabolites on the soluble form of NADP+:isocitrate dehydrogenase

The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by metabolites. Stopped flow kinetics showed a distinct lag time, followed by attainment of an apparently linear final velocity. Direct nonlinear regression analyses of the reaction progress curves allowed for the calculation of the rate constant (kappa) for the transition of the enzyme from an inactive to an active form; this transition is best catalyzed by its metal-substrate complex. Preincubation with metal-substrate or metal-citrate nearly abolished the lag by increasing kappa 10-fold. In steady state experiments, analyses of velocity versus metal-citrate complex as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, showed that binding of metal-citrate complex could both activate and inhibit the enzyme. This analysis suggested: (a) activation by binding to sites with an average dissociation constant of 0.25 mM; (b) inhibition by binding to sites with an average dissociation constant of 3.83 mM; and (c) modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-isocitrate serve to modulate the activity of NADP+:isocitrate dehydrogenase.     

Assignment of enzymatic functions to specific regions of the PLP-dependent heme protein cystathionine beta-synthase.

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyridoxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction involves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The enzyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase extending from residues 1 through 408 in which the C-terminal 143 residues have been deleted. This is similar to a natural variant of the protein that has been described in a homocystinuric patient in which the predicted peptide is 419 amino acids in length. Truncation leads to the formation of a dimeric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the absence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a K(d) of 7. 4 microM. Steady-state kinetic analysis indicates that the order of substrate addition is important. Thus, preincubation of the enzyme with homocysteine leads to a 2-fold increase in V(max) relative to preincubation of the enzyme with serine. Since the intracellular concentration of serine is significantly greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and homology searches with protein sequences in the database, we assign residues 68-209 as being important for PLP binding, residues 241-341 for heme binding, and residues 421-469 for AdoMet binding.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions

The hysteretic calmodulin-induced inactivation of muscle phosphofructokinase and the calmodulin-mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg . ATP, fructose 6-phosphate, adenosine 5'-[beta, gamma-imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate was studied by two techniques. (a) A two-step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions. Dominating in the interplay of calmodulin with metabolic effectors is the competitive-like action of calmodulin on Mg . ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg . ATP. In the allosteric assay it counteracts the ATP-induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a minor degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6-phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.     

Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms

In this study, an analysis of the transition between the inactive ("closed") and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein.     

Use of intrinsic binding energy for catalysis by a cofactor-independent DNA enzyme

The concept of the Circe effect, according to which an enzyme's substrate-binding energy is utilized to destabilize the substrate towards the reaction transition state, has been shown to be a relevant catalytic strategy for naturally occurring protein enzymes and for two ribozymes that use nucleotide-based substrates and metal ion cofactors. We wished to investigate whether such a catalytic strategy extends even to divergent and unevolved catalysts constructed from biopolymers. We examined the properties of a small, in vitro selected, and cofactor-independent DNA enzyme, PS5.M, which catalyzes porphyrin metallation. The metallation reaction is unique, in that the energies for binding and for metallation of both the substrate and of a transition-state analogue (TSA) can be measured. We report that PS5.M, originally selected for binding to the TSA, displays the Circe effect in channeling a significant component of entropy-rich "intrinsic" binding energy to distort and to alter the basicity of the bound substrate. The study demonstrates that nucleic acids are, by themselves, capable of creating active sites for the catalysis of chemical reactions involving non-nucleotide substrates. Furthermore, the study of the metallation of the TSA provides a quantitative estimate of the effectiveness of such a compound in mimicking the true transition state for porphyrin metallation.     

Tyrosinase exhibits lag at pH 6.8 under steady state concentrations of tyrosine and 3,4-dihydroxy phenyl alanine in melanoma tissue.

Tyrosine to dopa ratio determines the extent of lag in cresolase activity of tyrosinase when assayed at pH 6.8. The levels of tyrosine and dopa in B-16 murine melanoma tissue were found to be 213 and 13 mmoles/g fresh wt of tissue respectively. Cresolase activity of tyrosinase, when assayed at the above steady state levels of tyrosine and dopa at pH 6.8, exhibited a lag of 5-15 min depending on the amount of enzyme used in the assay mixture and the initial enzyme activity was zero. Under in vivo conditions, the enzyme with zero initial activity can not be active and therefore a far reaching conclusion is that tyrosine to dopa ratio may not regulate the enzyme activity, unlike under in vitro conditions. Possible modes of the regulation of tyrosinase under in vivo conditions are discussed.