Proteolysis mediated by the membrane‐integrated ATP‐dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates

ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane‐integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40–60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water‐soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.     

On the mechanism of SPP-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane

Intramembrane-cleaving proteases are members of a novel type of enzyme that hydrolyse substrate proteins within transmembrane regions. The presently known proteases that catalyse such cleavage reactions are membrane proteins of high hydrophobicity and multiple predicted transmembrane regions. A key feature is the positioning of active site residues in hydrophobic segments implying that the catalytic centre is assembled within the plane of the membrane. Nevertheless, all these proteases appear to utilise catalytic mechanisms similar to classic proteases that expose their active site domains in aqueous compartments. In the present review, we will address the mechanism of intramembrane proteolysis on the example of the signal peptide peptidase, and discuss how enzyme-catalysed hydrolysis of peptide bonds within the plane of a cellular membrane might occur.     

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.     

Hydrolysis of whey proteins by proteases extracted from Cynara cardunculus and immobilized onto highly activated supports

Blends of cardosins A and B, enzymes present in aqueous extracts of the flowers of the thistle (Cynara cardunculus L.), have for long been used as rennets by the cheesemaking industry in the Iberian Peninsula. These dimeric proteases are present in the stigmae and stylets of said flowers, and are thought to play a role in sexual reproduction of the plant. In the present research effort, production of cardosin derivatives (starting from a crude extract), encompassing full stabilization of their dimeric structure, has been attempted via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to leaching of enzyme, thus proving the effectiveness of the attachment procedure. Furthermore, derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at lab-scale trials to perform (selective) hydrolysis of alpha-lactalbumin, one of the major proteins in bovine whey.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Enzymatic hydrolysis of whey proteins. II. Molecular-weight range

Using high-pressure liquid chromatography we studied the distribution of molecular weights in whey-protein hydrolysates using the following commercially obtained proteases: Alcalasa 0.6 L and Protease 660 L, both bacterial in origin, and PEM 2500 S, of animal origin. In each of the systems, the range of molecular weights in the hydrolysate depended solely on the degree of hydrolysis (DH) achieved. For DH >/= 20, between 65% and 95% of the hydrolysate is made up of peptides with a molecular weight of less than 1,000 Da. (c) 1994 John Wiley & Sons, Inc.     

Process Optimization for the Production of Bio-functional Whey Protein Hydrolysates: Adopting Response Surface Methodology

Whey is a protein complex derived from milk, exhibit highest protein quality rating among other proteins, being touted as a functional food with number of health benefits. In the present investigation, whey proteins hydrolysates produced using trypsin enzyme to augment antioxidant activity and to assess angiotensin converting enzyme (ACE) inhibition activity. Hydrolysis parameters were standardized applying response surface methodology. The response antioxidant activity in terms of Trolox equivalent antioxidant capacity (TEAC) values was determined by radical scavenging assay method. Optimum conditions for maximum antioxidant activity were standardized at 88 °C of preheating, 7.3 pH, 0.05 enzymes to substrate ratio and hydrolysis was carried up to 8 h at 36.5 °C. Resulting peptide fractions obtained at 11.8 % of degree of hydrolysis displayed antioxidant capacity with TEAC values of 1.37 ± 0.12. The designed model found to be significant with R² value of 0.9972 for antioxidant activity and lack of fit test-as non significant, indicating that the optimized conditions were best suited. The hydrolysate further investigated for antihypertensive activity. The outcome indicate that to affect decrease in ACE inhibition activity 4,166.72 μg of native whey protein is required when compared to 229.96 μg of hydrolysates. These results indicate hydrolysate produced under these conditions could be an effective nutraceutical.     

ATP-dependent FtsH and Lon chloroplast proteases

Arabidopsis thaliana proteome contains 667 proteases; some tens of them are chloroplast-targeted proteins, encoded by genes orthologous to the ones coding for bacterial proteolytic enzymes. It is thought that chloroplast proteases are involved in chloroplasts' proteins turnover and quality control (maturation of nucleus-encoded proteins and removal of nonfunctional ones). Some ATP-dependent chloroplast proteases belonging to FtsH family (especially FtsH2 and FtsH5) are considered to be involved in numerous aspects of chloroplast and whole plant maintenance under non-stressing as well as stressing conditions. This notion is supported by severe phenotype appearance of mutants deficient in these proteases. In contrast to seemingly high physiological importance of chloroplast members of FtsH protease family, only a few individual proteins have been identified so far as their physiological targets (i.e. Lhcb1, Lhcb3, PsbA and Rieske protein). Our knowledge regarding structure and molecular mechanisms of these enzymes' action is limited when compared with what is known about FtsHs of bacterial origin. Equally limited is the knowledge about ATP-dependent Lon4 protease being the single known chloroplast-targeted ortholog of Lon protease of Escherichia coli.     

PACMANS: A bioinformatically informed algorithm to predict,design, and disrupt protease‐on‐protease hydrolysis

Multiple proteases in a system hydrolyze target substrates, but recent evidence indicates that some proteases will degrade other proteases as well. Cathepsin S hydrolysis of cathepsin K is one such example. These interactions may be uni‐ or bi‐directional and change the expected kinetics. To explore potential protease‐on‐protease interactions in silico, a program was developed for users to input two proteases: (1) the protease‐ase that hydrolyzes (2) the substrate, protease. This program identifies putative sites on the substrate protease highly susceptible to cleavage by the protease‐ase, using a sliding‐window approach that scores amino acid sequences by their preference in the protease‐ase active site, culled from MEROPS database. We call this PACMANS, Protease‐Ase Cleavage from MEROPS ANalyzed Specificities, and test and validate this algorithm with cathepsins S and K. PACMANS cumulative likelihood scoring identified L253 and V171 as sites on cathepsin K subject to cathepsin S hydrolysis. Mutations made at these locations were tested to block hydrolysis and validate PACMANS predictions. L253A and L253V cathepsin K mutants significantly reduced cathepsin S hydrolysis, validating PACMANS unbiased identification of these sites. Interfamilial protease interactions between cathepsin S and MMP‐2 or MMP‐9 were tested after predictions by PACMANS, confirming its utility for these systems as well. PACMANS is unique compared to other putative site cleavage programs by allowing users to define the proteases of interest and target, and can also be employed for non‐protease substrate proteins, as well as short peptide sequences.     

Chloroplast proteases: possible regulators of gene expression?

A wide range of proteolytic processes in the chloroplast are well recognized. These include processing of precursor proteins, removal of oxidatively damaged proteins, degradation of proteins missing their prosthetic groups or their partner subunit in a protein complex, and adjustment of the quantity of certain chloroplast proteins in response to changing environmental conditions. To date, several chloroplast proteases have been identified and cloned. The chloroplast processing enzyme is responsible for removing the transit peptides of newly imported proteins. The thylakoid processing peptidase removes the thylakoid-transfer domain from proteins translocated into the thylakoid lumen. Within the lumen, Tsp removes the carboxy-terminal tail of the precursor of the PSII D1 protein. In contrast to these processing peptidases which perform a single endo-proteolytic cut, processive proteases that can completely degrade substrate proteins also exist in chloroplasts. The serine ATP-dependent Clp protease, composed of the proteolytic subunit ClpP and the regulatory subunit ClpC, is located in the stroma, and is involved in the degradation of abnormal soluble and membrane-bound proteins. The ATP-dependent metalloprotease FtsH is bound to the thylakoid membrane, facing the stroma. It degrades unassembled proteins and is involved in the degradation of the D1 protein of PSII following photoinhibition. DegP is a serine protease bound to the lumenal side of the thylakoid membrane that might be involved in the chloroplast response to heat. All these peptidases and proteases are homologues of known bacterial enzymes. Since ATP-dependent bacterial proteases and their mitochondrial homologues are also involved in the regulation of gene expression, via their determining the levels of key regulatory proteins, chloroplast proteases are expected to play a similar role.     

Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process.

To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process.     

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in?vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.     

ClpP hydrolyzes a protein substrate processively in the absence of the ClpA ATPase: mechanistic studies of ATP-independent proteolysis

ATP-dependent proteases are processive, meaning that they degrade full-length proteins into small peptide products without releasing large intermediates along the reaction pathway. In the case of the bacterial ATP-dependent protease ClpAP, ATP hydrolysis by the ClpA component has been proposed to be required for processive proteolysis of full-length protein substrates. We present here data showing that in the absence of the ATPase subunit ClpA, the protease subunit ClpP can degrade full-length protein substrates processively, albeit at a greatly reduced rate. Moreover, the size distribution of peptide products from a ClpP-catalyzed digest is remarkably similar to the size distribution of products from a ClpAP-catalyzed digest. The ClpAP- and ClpP-generated peptide product size distributions are fitted well by a sum of multiple underlying Gaussian peaks with means at integral multiples of approximately 900 Da (7-8 amino acids). Our results are consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 (+/-2-3) amino acid residues and proteolysis. On the structural and molecular level, the step size may be controlled by the spacing between the ClpP active sites, and processivity may be achieved by coupling peptide bond hydrolysis to the binding and release of substrate and products in the protease chamber.     

Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds.

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.     

A serine alkaline protease from the fungusConidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.     

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.     

On the mechanism of alpha-amylase.

Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley alpha-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103-fold using rDP18-maltodextrin and 10(5) to 10(6)-fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. alpha-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.beta- and gamma-CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the alpha-amylase family.     

A C-terminal region of signal peptide peptidase defines a functional domain for intramembrane aspartic protease catalysis

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.     

Hydrolysis of α-lactalbumin by cardosin A immobilized on highly activated supports

In the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and β-mercaptoethanol did not lead to leaching of enzyme, thus providing evidence for the effectiveness of the attachment procedure. Furthermore, the cardosin A derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at laboratory-scale trials to perform (selective) hydrolysis of α-lactalbumin (α-La), one of the major proteins in bovine whey. Hydrolysates of α-La were assayed for by the OPA method, as well as by FPLC, SDS–PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also assessed at 40, 50 and 55 °C; at these temperatures, no thermal denaturation took place during incubation for 48 h. The highest degree of hydrolysis was attained by 5 h reaction, at 55 °C and pH 5.2. SDS–PAGE of α-La hydrolysates displayed bands corresponding to low molecular weight peptides. Our results suggest that cardosin A in immobilized form is a good candidate to bring about proteolysis in the dairy industry, namely in whey processing.