Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of amino acid residues involved in substrate and cation binding was investigated in complementary experiments on Fe(2+)-catalyzed oxidation and cleavage, limited digestion with proteinase K, and mutational analysis. Cleavage at Ser346 was produced by Fe(2+) in the presence of substrate (ATP or AMP-PNP) and Ca(2+), and was attributed to Fe(2+) bound to a Mg(2+) site near Ser346 and neighboring Glu696. Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain was also observed and attributed to Fe(2+) substituting for Mg(2+) in the Mg(2+)-ATP complex bound to the N domain. Mutation of Arg560 or Glu439 within the N domain interfered with nucleotide-dependent ATPase resistance to digestion with proteinase K. Furthermore, mutation of Lys352, Lys684, Thr353, Asp703, or Asp707 within the P domain produced similar interference, consistent with a role of these residues in substrate stabilization at the catalytic site. In a third group of experiments, equilibrium isotherms were obtained with Asn796Ala and Glu309Gln mutants, demonstrating non-cooperative binding of one Ca(2+) per ATPase, as opposed to cooperative binding of two Ca(2+) by WT enzyme. No high-affinity binding by Asp800Asn, Glu771Gln, and Thr799Ala mutants was detected. It was also demonstrated that the conformational transitions involved in enzyme activation and interconversion of Ca(2+) binding and phosphorylation energy, are triggered by Ca(2+) binding to site II and stabilization of Glu309 (M4) and N796 (M6).
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PMID:Characterization of Ca2+ ATPase residues involved in substrate and cation binding. 1276 76

After proteinase K-induced excision of five amino acid residues in the semiconserved polypeptide chain linking the end of the A domain with the S3/M3 transmembrane segment we find that Ca(2+) transport is blocked while partial reactions like Ca(2+) binding, ATP phosphorylation, and Ca(2+)-occlusion are left intact. However, formation of the so-called E2P state (either from the phosphorylated species formed in the presence of ATP and Ca(2+) or from the Ca(2+)-depleted unphosphorylated species) is blocked. We conclude that the proteinase K-treated ATPase, while maintaining many of the partial reactions, is incapable of energy transduction because of the absence of an E2P state with Ca(2+) binding sites exposed to the intravesicular space. Sequence comparisons and mutagenesis data point to an important role in energy transduction of P-type ATPases of a conserved motif located at the end of the A domain.
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PMID:Proteolytic studies on the transduction mechanism of sarcoplasmic reticulum Ca2+-ATPase: common features with other P-type ATPases. 1276 78

The bile acid intermediate 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxisomal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.
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PMID:ATP-dependent transport of bile acid intermediates across rat liver peroxisomal membranes. 1296 71

By measuring the phosphorylation levels of individual proteolytic fragments of SERCA1a separated by electrophoresis after their phosphorylation, we were able to study the catalytic properties of a p95C-p14N complex arising from SERCA1a cleavage by proteinase K between Leu(119) and Lys(120), in the loop linking the A-domain with the second transmembrane segment. ATP hydrolysis by the complex was very strongly inhibited, although ATP-dependent phosphorylation and the conversion of the ADP-sensitive E1P form to E2P still occurred at appreciable rates. However, the rate of subsequent dephosphorylation of E2P was inhibited to a dramatic extent, and this was also the case for the rate of "backdoor" formation of E2P from E2 and P(i). E2P formation from E2 at equilibrium nevertheless indicated little change in the apparent affinity for P(i) or Mg(2+), while binding of orthovanadate was weaker. The p95C-p14N complex also had a slightly reduced affinity for Ca(2+) and exhibited a reduced rate for its Ca(2+)-dependent transition from E2 to Ca(2)E1. Thus, disruption of the N-terminal link of the A-domain with the transmembrane region seems to shift the conformational equilibria of Ca(2+)-ATPase from the E1/E1P toward the E2/E2P states and to increase the activation energy for dephosphorylation of Ca(2+)-ATPase, reviving the old idea of the A-domain being a phosphatase domain as part of the transduction machinery.
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PMID:Functional properties of sarcoplasmic reticulum Ca(2+)-ATPase after proteolytic cleavage at Leu119-Lys120, close to the A-domain. 1467 56

Mycoplasma mobile glides on surfaces at up to 7 microm/s by an unknown mechanism. We studied the energetics that power gliding by using a novel, growth medium-free system. We found that cells could glide in defined media if the glass substrate is preconditioned by exposure to horse serum. The active component that potentiates gliding is sensitive to proteinase K treatment. We used the defined medium system to test the effect of various inhibitors, ionophores, and poisons on motility of M. mobile. Valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin, and puromycin had no short-term effects on gliding. We also confirmed that we were able to modulate the membrane potential with valinomycin and FCCP by using a potential-sensitive dye. Shifting the pH likewise had no effect on motility. These results rule out the use of conventional ion motive forces to power gliding. Arsenate had a dramatic inhibitory effect on gliding, and both the speed and the fraction of cells moving tracked ATP levels. Sodium orthovanadate had a slight but significant inhibitory effect on gliding. Taken together, these results suggest that the motor system of M. mobile is likely an ATPase or is directly coupled to an ATPase.
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PMID:Energetics of gliding motility in Mycoplasma mobile. 1520 28

We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).
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PMID:Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism. 1526 96

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.
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PMID:Posttranslational modification of 6-phosphofructo-1-kinase in Aspergillus niger. 1574 45

Trypanosoma brucei mitochondria possess a unique RNA decay pathway in which rapid degradation of polyadenylated mRNAs is dependent on the addition of UTP, as measured by in organello pulse chase assays. To determine the mechanism by which UTP stimulates the degradation of polyadenylated RNAs, we performed in organello pulse chase assays under different conditions. Treatment of mitochondria with proteinase K revealed that UTP does not act through a receptor on the surface of the mitochondria. To determine if the UTP-stimulated RNA decay pathway is triggered by the mitochondrial energy state or ATP:UTP ratio, increasing ATP was added to a constant amount of UTP during the chase period of the assay. Results indicate that rapid turnover is responsive to UTP and not the ATP:UTP ratio. Experiments using UTP analogs demonstrate that UTP polymerization into RNAs is necessary for UTP-dependent degradation. Furthermore, experiments performed with RNAi cells indicate that the RET1 terminal uridylyl transferase (TUTase) is required for UTP-dependent decay of polyadenylated RNAs. Overall, these results show that degradation of polyadenylated RNAs in T. brucei mitochondria can occur through a unique mechanism that requires the polymerization of UTP into RNAs, presumably by the RET1 TUTase.
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PMID:UTP-dependent turnover of Trypanosoma brucei mitochondrial mRNA requires UTP polymerization and involves the RET1 TUTase. 1581 18

Quantitation of cellular adenylate levels (i.e., ATP, ADP, AMP) has widespread applications in physiological, metabolic and energetic studies. We have compared classical adenylate extraction procedures (i.e., perchloric acid, boiling) with a previously unreported proteinase K-based extraction technique. Our results suggest that all three techniques are comparable in soft animal tissue, but proteinase K-based extractions consistently generated higher adenylate yields from a broad range of organisms, particularly those containing a cell wall (e.g., alga, bacteria, fungi, plant).
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PMID:Quantitating adenylate nucleotides in diverse organisms. 1589 79

Treatment of an N-terminal-containing His6-tagged insulysin (His6-IDE) with proteinase K led to the initial cleavage of the His tag and linker region. This was followed by C-terminal cleavages resulting in intermediate fragments of approximately 95 and approximately 76 kDa and finally a relatively stable approximately 56 kDa fragment. The approximately 76 and approximately 56 kDa fragments exhibited a low level of catalytic activity but retained the ability to bind the substrate with a similar affinity as the native enzyme. The kinetics of the reaction of the IDE approximately 76 and approximately 56 kDa proteolytic fragments with a synthetic fluorogenic substrate produced hyperbolic substrate versus velocity curves, rather than the sigmoidal curve obtained with His6-IDE. The approximately 76 and approximately 56 kDa IDE proteolytic fragments were active toward the physiological peptides beta-endorphin, insulin, and amyloid beta peptide 1-40. Although activity was reduced by a factor of approximately 103-104 with these substrates, the relative activity and the cleavage sites were unchanged. Both the approximately 76 and approximately 56 kDa fragments retained the regulatory cationic binding site that binds ATP. Thus, the two proteinase K cleavage fragments of IDE retain the substrate- and ATP-binding sites but have low catalytic activity and lose the allosteric kinetic behavior of IDE. These data suggest a role of the C-terminal region of IDE in allosteric regulation.
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PMID:Proteolytic fragments of insulysin (IDE) retain substrate binding but lose allosteric regulation. 1715 46


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