Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. II. Purification and characterization of the enzyme

Prephenoloxidase-activating enzyme has been purified approximately 4800-fold from cuticular extract of the silkworm, and the preparation seems to be homogeneous as judged by disc- and dodecylsulfate-polyacrylamide gel electrophoresis. By means of gel filtration through Sephadex G-100, it has been supposed that the enzyme exists as mono- and dimeric forms at slightly acidic pH, while a monomeric form is predominant under slightly alkaline condition. The molecular weight of the monomer was estimated to be 33,000–35,000 by dodecylsulfate-polyacrylamide gel electrophoresis and gel filtration.It has been demonstrated that ester substrates for trypsin, benzoyl-l-arginine ethyl ester and tosyl-l-arginine methyl ester, can be hydrolyzed by the purified enzyme. Several lines of evidence indicating that a single protein is involved in both activation and esterolytic reactions have been presented. Some enzymatic properties of the purified preparation as esterase have also been described.In connection to esterase activity of the purified enzyme, a mechanism of prephenoloxidase activation in the silkworm system has briefly been discussed.     

Activation of prephenoloxidase. 3. Release of a peptide from prephenoloxidase by the activating enzyme

Activation reaction of prephenoloxidase (pre-enzyme) was analyzed using a system of homogeneous pre-enzyme and highly purified prephenoloxidase-activating enzyme (PPAE) from the silkworm $\text{Bombyx mori}$. When pre-enzyme was activated by PPAE, release of a peptide was demonstrated. Results of polyacrylamide gel electrophoresis revealed that only a single peptide is liberated from pre-enzyme. Several lines of evidence indicated that the released peptide is inhibitory on PPAE.     

Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme.

NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.     

Specific ligand-induced association of an enzyme. A new model of dissociating allosteric enzyme

The kinetic behavior of dissociative enzyme system of the type inactive monomer in equilibrium active dimer where dimeric form is stabilized by specific ligand (in particular by substrate) which is bound in the region of the contact of monomers has been analysed. It is assumed that the dissociation of dimer results in formation of monomers which retain the subsites for specific ligand binding. The shape of the dependences of enzyme reaction rate (v) on substrate concentration (S) has been characterized using the order of enzyme reaction rate with respect to substrate concentration: ns = d ln v/d ln [S]. When the substrate concentrations are low the dependences of v on [S] have S-shaped form (the maximum value of ns exceeds the unity) at the definite values of the parameters of the enzyme system. The value of ns approaches--2 at sufficiently high substrate concentrations (in the region where the substrate reveals the inhibitory effect due to blocking the association of inactive monomers into active dimer). The methods of calculation of the parameters of the dissociative enzyme system under discussion have been elaborated on the basis of the analysis of the experimental dependences of specific enzyme activity on enzyme concentration obtained at various fixed substrate concentrations.     

Conversion of p-coumarate into caffeate by Streptomyces nigrifaciens. Purification and properties of the hydroxylating enzyme

1. An enzyme responsible for the conversion of p-coumarate into caffeate was purified 97-fold from Streptomyces nigrifaciens. The enzyme had a molecular weight of 18000 as determined by Sephadex G-100 gel filtration and was homogeneous on polyacrylamide-gel electrophoresis. 2. The preparation exhibited both p-coumarate hydroxylase and caffeate oxidase activities. 3. Stoicheiometry of the reaction indicated a mono-oxygenase-mediated catalysis consuming 1mol of O(2)/mol of substrate hydroxylated. 4. NADH, NADPH, tetrahydropteroylglutamate or ascorbate act as electron donors for the reaction, ascorbate being inhibitory at higher concentrations. 5. The optimum enzyme activity was at about pH7.7 and 40 degrees C, with an activation energy of 39kJ/mol. 6. Monophenols such as p-hydroxyphenylpropionate, p-hydroxyphenylacetate, l-tyrosine and dl-p-hydroxyphenyl-lactate were also hydroxylated by the preparation, in addition to p-coumarate. 7. The enzyme was a copper protein having 0.38% copper in a bound form. 8. Thiol-group inhibitors did not affect the reaction. 9. The relationship of the enzyme to other hydroxylases is discussed.     

Mechanisms of enzyme action and inhibition: Transition state analogues for acid-base catalysis

The theory of absolute reaction rates implies that the grip of a catalyst on a substrate tightens with substrate activation, relaxing later as products are formed and released. Analogs that mimic different kinds of substrate activation can, through the structural details of their complexes with enzymes, indicate how active site residues are involved in the enhancement of reactions rates. In several cases, bonds involved in general acid-base catalysis have been identified tentatively; and recent evidence points to a hydrogen bond of remarkable stability in the transition state in enzymatic deamination of adenosine. Similar approaches have been used to enzymes that act primarily by substrate distortion, nucleophilic catalysis, solvent removal and catalysis by approximation. Two recurring observations, that were not expected in theory, have been the binding of inhibitors in ionized forms that are rare in solution, and changes in enzyme configuration that accompany binding of transition state analogs. Origins and implications of these findings will be discussed with specific reference to the role of solvent water in catalytic phenomena.Supported by Grant GM-18325 from the National Institutes of Health.     

Temperature dependence of a diffusion-limited immobilized enzyme reaction.

The apparent activation energy of N-alpha-benzoyl-L-arginine-ethyl ester (BAEE) hydrolysis by immobilized trypsin varies with the bulk substrate concentration from its maximum value, comparable to that of the free enzyme, to considerably lower values. Thus, with a concentration change from 3 x 10(-2) to 10(-4) M the apparent activation energy diminishes from 9.5 to 4.5 kcal/mol. This experimental finding is interpreted to be due to Michaelis-type kinetics in a heterogeneous system, in one case reflecting the temperature dependence of the maximal enzyme reaction rate, in another case illustrating the diffusion limited overall reaction at low substrate concentrations. As a consequence it may not be feasible to operate a reaction at elevated temperatures in a high conversion range, since diffusion limitation may restrict the enhancement of the overall reaction rate. Some further data are given concerning the buffer effect on the reaction rate, which should occur due to its limitation by proton transfer in the buffer-free system.     

Isopentenyl pyrophosphate isomerase:dimethylallyl pyrophosphate isomerase: isolation from Claviceps sp. SD 58 and comparison to the mammalian enzyme

Isopentenyl pyrophosphate isomerase:dimethyl pyrophosphate isomerase (EC 5.3.3.2) has been purified to near homogeneity from Claviceps sp. A molecular weight of 35,000 was found by gel exclusion chromatography as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the enzyme consists of a single subunit and is in contrast to the Mr 22,000 that we have found for the enzyme from liver. The lability of isomerase from liver, often reported, has been found to be due to its susceptibility to proteolysis. Nine compounds have been tested as inhibitors of both isomerases. The binding of analogs requires the pyrophosphate moiety which may be substituted by a variety of alkyl groups. Inclusion of a polar function in the hydrocarbon portion of the analog greatly reduces interaction with the enzyme. Reversibility of the reaction was not found with a higher homolog of the substrate.     

Mechanism of the inhibitory effect of glyoxylate plus oxaloacetate and oxalomalate on the NADP-specific isocitrate dehydrogenase.

The effects of glyoxylate plus oxaloacetate and of oxalomalate on the NADP-linked isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating, EC 1.1.1.42) from pig heart from been studied with steady state methods as well as with stopped flow technique. When equimolar mixtures of glyoxylate and oxaloacetate were premixed for different lengths of time prior to addition to the assay mixture, the extent of inhibition increased with the premixing time. The results indicated that the inhibition by glyoxylate plus oxaloacetate is caused by a compound formed in a reversible interaction between the two components. Glyoxylate plus oxaloacetate and oxalomalate affected the enzyme in at least three different ways. They inhibited the enzyme in a reaction competitive with regard to the substrate isocitrate. This inhibition needed a certain time to be fully expressed. The time lag could be eliminated by premixing of the enzyme and inhibitor with NADP plus metal ion. Secondly, if the enzyme is premixed with NADP plus metal ions, a time lag occurs before the reaction rate approaches a constant value after initiation of the reaction with isocitrate. The inhibitors were found to enhance this effect of NADP plus metal ions on the enzyme. Thirdly, it has previously been shown that the enzyme can be activated by metal complexing agents. Glyoxylate plus oxaloacetate as well as oxalomalate are able to form complexes with metal ions and were found to cause an initial activation of the enzyme under certain assay conditions. The controversy regarding the mechanism of action of the above inhibitors on the enzyme is probably due to the fact that they affect the enzyme in several different ways.     

Mechanism of action of cyclic AMP-dependent histone kinase. Substrate specificity of the catalytic enzyme unit]

Interaction of several nucleotide derivates with homogenous catalytic subunit of cyclo-AMP-dependent histone kinase from pig brain is studied. Inhibition constants of these compounds are calculated, and the affinity of inhibitors to the enzyme active site is evaluated. The nature of heterocyclic base is found to be the main contribution into binding with substrate. The enzyme specificity with respect to a number of bivalent metal ions is studied, and Mg2+ is demonstrated to be the only efficient enzyme activator. It is shown by means of stationary kinetics that histone kinase-catalysed phosphotransferase reaction has a "ping-pong"-like mechanism.     

Thermodynamic and extrathermodynamic requirements of enzyme catalysis

An enzyme's affinity for the altered substrate in the transition state (symbolized here as S) matches the value of k(cat)/K(m) divided by the rate constant for the uncatalyzed reaction in water. The validity of this relationship is not affected by the detailed mechanism by which any particular enzyme may act, or on whether changes in enzyme conformation occur on the path to the transition state. It subsumes potential effects of substrate desolvation, H-bonding and other polar attractions, and the juxtaposition of several substrates in a configuration appropriate for reaction. The startling rate enhancements that some enzymes produce have only recently been recognized. Direct measurements of the binding affinities of stable transition-state analog inhibitors confirm the remarkable power of binding discrimination of enzymes. Several parts of the enzyme and substrate, that contribute to S binding, exhibit extremely large connectivity effects, with effective relative concentrations in excess of 10(8) M. Exact structures of enzyme complexes with transition-state analogs also indicate a general tendency of enzyme active sites to close around S in such a way as to maximize binding contacts. The role of solvent water in these binding equilibria, for which Walter Kauzmann provided a primer, is only beginning to be appreciated.     

Active enzyme gel chromatography: II. Computer simulations

The behavior of an enzyme undergoing reaction while on a gel chromatography column has been studied by computer simulation using the steady state assumption for a system with a single enzyme—substrate complex. The profiles of the enzyme—substrate complex, product, and substrate were examined varying the parameters of k_cat, flow rate, partition coefficient, dispersion coefficient, and time. These investigations confirm that much information about both the active enzyme and the product may be obtained by examining the product profile alone, varying the power of applying scanning gel chromatography to active enzyme systems.     

Kinetic studies of L-aspartase from Escherichia coli: pH-dependent activity changes

The pH dependence of the kinetic parameters of the L-aspartase-catalyzed reaction have been examined in both the amination and the deamination directions. The enzyme isolated from Escherichia coli exists in a pH-dependent equilibrium between a higher pH form that has an absolute requirement for a divalent metal ion and for substrate activation, and a low pH form that does not require activation by either substrate or metal ions. The interconversion between these enzyme forms is observed near neutral pH in the profiles examined for the reaction in either direction. This pH-dependent activation has not been observed for other bacterial aspartases. Loss of activity is observed at high pH with a pK value of 9. The pH profiles of competitive inhibitors such as 3-nitropropionic acid and succinic acid have shown that the enzyme group responsible for this activity loss must be protonated for substrate binding at the active site. An enzymatic group has also been identified that must be protonated in the amination reaction, with a pK value near 6.5, and deprotonated in the deamination reaction. This group, tentatively assigned as a histidyl residue, fulfills the criteria for the acid-base catalyst at the active site of L-aspartase.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Elucidation of enzyme mechanisms using fluorinated substrate analogues

A great variety of biological reactions that are physiologically important are catalyzed by enzymes. Understanding the reaction course of these enzyme-catalyzed transformations are of significant importance since the insights gained from these experiments may facilitate the design of methods to control or mimic their actions. A common strategy to study enzyme catalyses is to use fluorinated substrate analogues as mechanistic probes, since fluorine is an effective hydroxyl group mimic and can also be used to replace a hydrogen atom. Using fluorinated substrate probes have enabled researchers to obtain crucial information regarding the catalytic mechanism of enzymatic reactions. Many of these compounds are good enzyme inhibitors and have been developed into clinically useful chemotherapeutic agents. This review will discuss some examples of the use of fluorine containing compounds as mechanistic probes/enzyme inhibitors, many of which are selected from our own work.     

High pressure enzyme kinetics of dextransucrase.

The pressure dependence of enzymatic dextran formation has been observed up to 1000 at for several substrate concentrations. First order denaturation effects could be separated from the thermodynamic effects, which lead to a volume of 30.4 to 44.0 ccm per mole for the formation and -13.6ccm per mole for the activation of the enzyme-substrate complex. Denaturation depends on the substrate concentration. This leads to the conslusion that only the free enzyme is denatured, wheras the ES complex is stable.     

Dynamic aspects of enzyme specificity.

There are two aspects of enzyme specificity: recognition of the substrate by the formation of an enzyme-substrate compound and recognition of the transition state by catalysis of the reaction. Kinetic studies with inactive substrate analogues as potential competitive inhibitors, and structural studies of their compounds with enzymes, give information about the first of these specificity elements. Comparative kinetic studies with alternative substrates give information about both. There is a great deal of information from kinetic studies of dehydrogenases about the coenzyme specificities, substrate specificities and stereospecificities and mechanisms of these enzymes, particularly alcohol dehydrogenases. Recent X-ray diffraction studies of dehydrogenases have given insight into the molecular basis of some of their specificity elements. An attempt is made to correlate the available kinetic and structural data for alcohol and lactate dehydrogenases.     

Procarboxypeptidase A from the insect pest Helicoverpa armigera and its derived enzyme. Two forms with new functional properties.

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine-specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C-terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a Ki in the pm range.     

A versatile equation to describe reversible enzyme inhibition and activation kinetics: modeling beta-galactosidase and butyrylcholinesterase

Current treatments for Alzheimer's disease involve inhibiting cholinesterases. Conversely, cholinesterase stimulation may be deleterious. Homocysteine is a known risk factor for Alzheimer's and vascular diseases and its active metabolite, homocysteine thiolactone, stimulates butyrylcholinesterase. Considering the opposing effects on butyrylcholinesterase of homocysteine thiolactone and cholinesterase inhibitors, understanding how these molecules alter this enzyme may provide new insights in the management of dementia. Butyrylcholinesterase does not strictly adhere to Michaelis-Menten parameters since, at higher substrate concentrations, enzyme activation occurs. The substrate activation equation for butyrylcholinesterase does not describe the effects of inhibitors or non-substrate activators. To address this, global data fitting was used to generate a flexible equation based on Michaelis-Menten principles. This methodology was first tested to model complexities encountered in inhibition by imidazole of beta-galactosidase, an enzyme that obeys Michaelis-Menten kinetics. The resulting equation was sufficiently flexible to permit expansion for modeling activation or inhibition of butyrylcholinesterase, while accounting for substrate activation of this enzyme. This versatile equation suggests that both the inhibitor and non-substrate activator examined here have little effect on the substrate-activated form of butyrylcholinesterase. Given that butyrylcholinesterase inhibition can antagonize stimulation of this enzyme by homocysteine thiolactone, cholinesterase inhibition may have a role in treating Alzheimer and vascular diseases related to hyperhomocysteinemia.