Inactive Catalytic Subunit Research Articles

Sensing the Generation of Intracellular Free Electrons Using the Inactive Catalytic Subunit of Cytochrome P450s as a Sink.

Cytochrome P450 reductase (CPR) abstracts electrons from Nicotinamide adenine dinucleotide phosphate H (NADPH), transferring them to an active Cytochrome P450 (CYP) site to provide a functional CYP. In the present study, a yeast strain was genetically engineered to delete the endogenous CPR gene. A human CYP expressed in a CPR-null (yRD−) strain was inactive. It was queried if Bax—which induces apoptosis in yeast and human cells by generating reactive oxygen species (ROS)—substituted for the absence of CPR. Since Bax-generated ROS stems from an initial release of electrons, is it possible for these released electrons to be captured by an inactive CYP to make it active once again? In this study, yeast cells that did not contain any CPR activity (i.e., because the yeasts’ CPR gene was completely deleted) were used to show that (a) human CYPs produced within CPR-null (yRD-) yeast cells were inactive and (b) low levels of the pro-apoptotic human Bax protein could activate inactive human CYPs within this yeast cells. Surprisingly, Bax activated three inactive CYP proteins, confirming that it could compensate for CPR’s absence within yeast cells. These findings could be useful in research, development of bioassays, bioreactors, biosensors, and disease diagnosis, among others.

A role for protein kinase CK2 in plant development: evidence obtained using a dominant‐negative mutant

Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed of two distinct subunits, alpha (catalytic) and beta (regulatory), that combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss of function of CK2 on plant development, we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open reading frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not caused by disruption of the light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of the G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion, with compelling effects on Arabidopsis development.

Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase

Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.

Both Isoforms of the cAMP-dependent Protein Kinase Catalytic Subunit Can Activate Transcription of the Prolactin Gene

Whereas molecular cloning experiments have provided evidence for the presence of two closely related genes for the catalytic subunit of the cAMP-dependent protein kinase, the possible interchangeability of these isoforms in initiating biological processes has not been examined. To test the role of the two forms of the kinase in regulating transcription, expression vectors containing the coding sequence of either kinase have been cotransfected with a fusion gene containing the prolactin promoter coupled to an appropriate marker gene. The results demonstrate that expression vectors for both isoforms of the catalytic subunit are able to increase prolactin promotor activity in a dose-dependent manner. The effects can be observed by measuring either marker gene activity or RNA levels. Transfection of an expression vector encoding an inactive catalytic subunit did not stimulate prolactin promoter activity. The results provide additional evidence for the role of the catalytic subunit of the cAMP-dependent kinase in mediating regulation of specific gene transcription and demonstrate that both forms of the catalytic subunit are capable of participating in the regulation of transcription.

Phosphorylase phosphatase. Comparison of active forms using peptide substrates.

Phosphorylase phosphatase isolated from rabbit skeletal muscle can be activated in several ways. Trypsin-Mn2+ treatment of the purified Mr = 70,000 complex or addition of Mn2+ alone to the isolated inactive catalytic subunit gives enzyme species that readily dephosphorylate phosphorylase a and the type 2 regulatory subunit of cAMP-dependent protein kinase as well as synthetic phosphopeptides corresponding to the phosphorylation sites of these proteins. In contrast, enzyme activated by phosphorylation of the regulatory subunit using factor FA (glycogen synthase kinase 3) and Mg2+-ATP and thought to be of physiological significance dephosphorylates the protein substrates but not the phosphopeptides. Likewise, the active catalytic subunit isolated following FA treatment could not act on the peptides unless Mn2+ ions (maximal effect at 250 microM) were added. Mg2+ and Ca2+ could not substitute for Mn2+. Such differences in substrate specificity are not seen with p-nitrophenyl phosphate which is dephosphorylated by all forms of the phosphatase. The results suggest that the primary sequence surrounding the phosphorylation site of the substrate is not all that is necessary for recognition by the FA-activated form of the enzyme. They are interpreted in terms of constraints within the enzyme that are relaxed following exposure to Mn2+ or by the additional determinants present in larger protein substrates.

Phosphorylase phosphatase from skeletal muscle membranes.

Microsomes containing 12-15 U/mg phosphorylase phosphatase were obtained from skeletal muscle glycogen particles following glycogen digestion and differential centrifugation. The phosphatase associated with the membranes is in an inhibited state; dilution induces dissociation and deinhibition of the enzyme. Phosphatase-depleted membranes can rebind purified phosphatase catalytic subunit but not the complex between catalytic subunit and inhibitor 2. Binding involves a receptor, deduced from saturation phenomena, which is responsible for inhibition of the bound enzyme and which is a protein, since trypsin treatment releases all bound enzyme and prevents rebinding. The phosphatase extracted from the membranes is of type 1 and is a mixture of complexes, the major ones displaying a Mr of 300,000 and 70,000. From these complexes the 35-kDa catalytic subunit can be obtained either by trypsin treatment or by acetone precipitation. Purification to homogeneity involves chromatography on polylysine and FPLC chromatography on Mono Q and Polyanion SI columns. The purified enzyme exhibits a specific activity of 26,800 U/mg (27,900 U/mg after trypsin treatment) and consists of a major protein of 38 kDa (SDS gel electrophoresis). A minor component of 33 kDa, which may represent either a proteolytic product or an isozyme, can be separated. Both 38-kDa and 33-kDa catalytic subunits form a 70-kDa inactive complex with inhibitor 2 and upon incubation of the complexes the catalytic subunit is slowly converted to the inactive conformation which can then be reactivated by either the kinase FA or trypsin and Mn2+. Alternatively the inactive catalytic subunit is reactivated by Mn2+ alone once it has been isolated by FPLC chromatography on SI. The observation that the same catalytic subunit is present at various cell locations (namely cytosol, glycogen particles and microsomes), though in different conformations, is in favour of the hypothesis that displacement of the catalytic subunit from one cell site to the other may represent a new mechanism for phosphatase regulation in skeletal muscle.

The ATP,Mg-dependent phosphatase: Role of Mg ions in the expression of the phosphorylase phosphatase activity

The activation of the ATP,Mg-dependent phosphatase [F CM] by kinase F A has been shown to involve the phosphorylation or thiophosphorylation of the modulator subunit [M] and the consequent isomerization of the catalytic subunit [F C] into the active conformation. The inactive catalytic subunit [free F C] exhibits substantial activity in the presence of non-physiological concentrations of Mn ions whereas the Mn 2+-activation of the intact F CM-enzyme requires the proteolytic destruction of the modulator subunit. The present study points to the importance of Mg 2+ in the activation of the phosphatase. The inactive catalytic unit can be activated by millimolar concentrations of Mg 2+ and the thiophosphorylated F CM-enzyme only expresses its phosphorylase phosphatase activity after a subsequent trypsin treatment in the presence of Mg ions.

Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit.

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.

Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin.

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM were found to have values of 10 microM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

Cooperative interactions in aspartate transcarbamoylase. 1. Hybrids composed of native and chemically inactivated catalytic polypeptide chains.

Hybrids of aspartate transcarbamoylase (EC 2.1.3.2; carbamoylphosphate: L-aspartate carbamoyltransferase) from Escherichia coli containing native (active) and pyridoxylated (inactive) catalytic polypeptide chains were constructed by a procedure involving the reversible acylation of amino groups with 3,4,5,6-tetrahydrophthalic anhydride. This technique exploited the charges contributed by the tetrahydrophthaloyl groups as a "chromatographic handle" for separating the various species. Enzyme-like molecules containing one fully active and one inactive catalytic subunit showed cooperative kinetic behavior, considerable inhibition by CTP, and a substantially increased apparent K(m) compared to the native enzyme. Similar properties were observed for an intrasubunit hybrid containing one inactive catalytic polypeptide chain in each subunit. The cooperative inter-and intra-subunit hybrids also exhibited conformational changes similar to those found for the native enzyme upon the addition of stereospecific ligands. These observations, taken together with data for other complexes of catalytic and regulatory subunits, illustrate the importance of the architecture of aspartate transcarbamoylase and the quaternary constraint stemming from the subunit interactions.