The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase K(M) and decrease V(max) for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine.     

Nucleotide-dependent substrate recognition by the AAA+ HslUV protease

ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.     

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.     

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.     

Kinetics of endosulfan degradation by Phanerochaete chrysosporium

The chlorinated pesticide, endosulfan, could be degraded by Phanerochaete chrysosporium under non-ligninolytic conditions, and this did not require direct contact with mycelium. The major metabolites formed were endosulfan sulfate and endosulfan diol. The rate of degradation depended on the initial concentration. With 2.5 mg endosulfan l^–1, degradation was at 0.23 mg l^–1 day^–1. The degradation could be described using a nonlinear rate expression that was similar to the Michaelis–Menten equation.     

Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

Kinetics of pentachlorophenol degradation by a Flavobacterium species

The kinetics of pentachlorophenol (PCP) degradation by a Flavobacterium sp. ATCC 39723 has been investigated. Sodium glutamate was supplied as an additional carbon source to increase the rate of cell growth and PCP degradation. A kinetic model including PCP toxicity for cell growth and PCP inhibition of its own degradation was developed. This model was also applied to 2,4-dichlorophenol (DCP) degradation by the same organism. Although PCP and DCP are degraded by different pathways, the model describes these two degradation processes very well.     

Kinetics of hydroperoxide degradation by NADP-glutathione system in mitochondria

Hydroperoxide decomposition by the NADP-glutathione system in rat liver mitochondria was analyzed. Mitochondria were found to contain high concentrations of the reduced form of glutathione (GSH) (4.32 +/- 0.50 nmol/mg) and NADPH (4.74 +/- 0.64 nmol/mg), and high activities of glutathione peroxidase and reductase. In the initial phase of the reaction, the rate of hydroperoxide decomposition was proportional to both the GSH level and the activity of GSH peroxidase. However, in the later steady state, the step of NADP reduction was rate-limiting, and the overall reaction rate was independent of the initial concentration of GSH, and activities of glutathione peroxidase and reductase. Some GSH was released from mitochondria during incubation, but the rate of the decomposition could be simply expressed as kappa [GSH]/2, where kappa is the first-order rate constant of the peroxidase and [GSH] is the intramitochondrial level of GSH in the steady state. The rate of the reaction in the steady state was also dependent on the NADPH level, its reciprocal being linearly correlated with [NADPH]-1. The rate of decomposition of hydroperoxide was influenced by the respiratory state. During state 3 respiration, the rate was greatly depressed, but was still considered to exceed by far the rate of physiological generation of hydroperoxide.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine.

ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.     

Serum protein degradation by hypochlorite

The structural integrity of serum proteins: albumin, immunoglobulin G, transferrin, ceruloplasmin and superoxide dismutase, and the functional activity of the latter two enzymes after their interaction with hypochlorite were studied. It was shown that the interaction between the proteins and hypochlorite resulted in protein injury and degradation of their native structure. In the case of ceruloplasmin and transferrin, a practically complete protein "dissipation" occurred, the albumin and superoxide dismutase structures being injured in a lesser degree. The inactivation of ceruloplasmin was slower than that of superoxide dismutase. The protein degradation by hypochlorite seems to be the main factor restricting the ability of the proteins to act as antiinflammatory drugs.     

Effect of substrate nitrogen on lignin degradation by Pleurotus ostreatus

In order to determine the effect of substrate nitrogen (N) on ligninolytic activity, Pleurotus ostreatus was grown in solid media containing either growth-limiting (1mM) or excess (10mM) NH₄Cl. After 25 days, ¹⁴C–CO₂ production from ¹⁴C-cornstover lignin in low-N medium was 3 times that in nitrogen (N)-rich medium. Supplementation of low-N medium with glucose (0.3%) further enhanced ligninolytic activity. Decolorization of an aromatic, polymeric dye, Poly R-481, in solid media was also greatest under N-limiting conditions.     

Asymmetric nucleotide transactions of the HslUV protease

ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA ⁺ machine containing one or two HslU₆ ATPases and the HslV₁₂ peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp.

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG.     

Kinetics of methane formation by granular sludge at low substrate concentrations

Summary Apparent K _m values for methanogenesis from volatile fatty acids by various sludge granules were determined from progress curve experiments. The influence of mass transfer resistance on the kinetics of methane formation in biofilms was demonstrated with the apparent K _m values thus obtained. It was shown that the effects of mass transfer resistance depend on the maximum specific activity of the biolayers and on their thickness. the data indicate that mass transfer resistance in methanogenic biolayers becomes only of significance at low substrate concentrations and in thick biolayers with high methanogenic activities.     

Kinetics of low molecular weight substrate hydrolysis by immobilized trypsin]

The catalytic properties of trypsin immoblized on silochrome were studied in a flow reactor with replacement. The hydrolysis of methyl ester N-n-tosyl-L-arginine obeys the Michaelis--Menten kinetics. The apparent K'm value for the system with immobilized trypsin is considerably lower than for native trypsin. The K'm value was decreased with an increase in the rate of the substrate flow through the reactor or when smaller-sized silochrome granules were used. It is assumed that the apparent K'm value for the immobilized system is due to diffusion. The effects of diffusion on the catalytic properties of the immobilized enzyme were estimated.