An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Sequence of a cDNA clone encoding pig heart mitochondrial CoA transferase.

We have isolated a full-length cDNA clone encoding the cytoplasmic precursor to pig heart mitochondrial CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase (3-oxoacid CoA transferase, EC 2.8.3.5], a key enzyme for ketone body catabolism. The deduced amino acid sequence indicates the presence of a 39-residue mitochondrial signal sequence and a 481-residue mature protein of molecular weight 52,197. CoA transferase is known to be susceptible to proteolytic cleavage to produce a nicked but active enzyme. We have identified the site of proteolysis, and analysis of the sequence in its vicinity suggests that the polypeptide may fold into two domains connected by a highly hydrophilic bridge.     

Two-stage autolysis of the catalytic subunit initiates activation of calpain I

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.     

Identification of major autolytic cleavage sites in the regulatory subunit of vascular calpain II. A comparison of partial amino-terminal sequences to deduced sequence from complementary DNA

Purified calpain II from vascular smooth muscle is a heterodimer consisting of catalytic (Mr = 76,000) and regulatory (Mr = 30,000) subunits. In the presence of Ca2+, the regulatory subunit undergoes stepwise autolysis resulting in enzyme activation. By slowing autoproteolysis, we identified major autolytic intermediates of the regulatory subunit. Gas-phase sequencing of the regulatory subunit and its autolytic fragments revealed that the NH2-terminus of the Mr = 30,000 form was blocked, whereas each fragment yielded a unique amino acid sequence, suggesting that autolysis proceeds in an NH2- to COOH-terminal direction. By comparison of actual amino acid sequences of autolytic cleavage intermediates to the full sequence deduced from cDNA, we have identified the major autolytic cleavage sites. Three different peptide bonds were cleaved, with neutral amino acids predominating on both sides of the peptide bond hydrolyzed. Importantly, leucine or isoleucine was identified in the second position upstream from the cleavage site in all three autolytic sequences. The presence of an upstream leucine residue in the autolytic cleavage sequence is reminiscent of the structure of potent microbial and synthetic peptide inhibitors of calpain.     

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.     

Isolation and characterization of a haploid germ cell-specific novel complementary deoxyribonucleic acid; testis-specific homologue of succinyl CoA:3-Oxo acid CoA transferase

We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.     

Structure of the mammalian CoA transferase from pig heart

Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme.     

Nucleolin is a calcium-binding protein

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.     

Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp

As reported previously [Parsonage, D., Luba, J., Mallett, T. C., and Claiborne, A. (1998) J. Biol. Chem. 273, 23812-23822], the flavoprotein alpha-glycerophosphate oxidases (GlpOs) from a number of enterococcal and streptococcal sources contain a conserved 50-52 residue insert that is completely absent in the homologous alpha-glycerophosphate dehydrogenases. On limited proteolysis with trypsin, the GlpO from Streptococcus sp. (m = 67.6 kDa) is readily converted to two major fragments corresponding to masses of approximately 40 and 23 kDa. The combined application of sequence and mass spectrometric analyses demonstrates that the 40-kDa fragment represents the N-terminus of intact GlpO (Met1-Lys368; 40.5 kDa), while the 23-kDa band represents a C-terminal fragment (Ala405-Lys607; 22.9 kDa). Hence, limited proteolysis in effect excises most of the GlpO insert (Ser355-Lys404), indicating that this represents a flexible region on the protein surface. The active-site and other spectroscopic properties of the enzyme, including both flavin and tryptophan fluorescence spectra, titration behavior with both dithionite and sulfite, and preferential binding of the anionic form of the oxidized flavin, were largely unaffected by proteolysis. Enzyme-monitored turnover analyses of the intact and nicked streptococcal GlpOs (at [GlpO] approximately 10 microM) demonstrate that the single major catalytic defect in the nicked enzyme corresponds to a 20-fold increase in K(m)(Glp); the basis for this altered kinetic behavior is derived from an 8-fold decrease in the second-order rate constant for reduction of the nicked enzyme, as measured in anaerobic stopped-flow experiments. These results indicate that the flexible surface region represented by elements of the GlpO insert plays an important role in mediating efficient flavin reduction.     

Catalytic Turnover-Based Phage Selection for Engineering the Substrate Specificity of Sfp Phosphopantetheinyl Transferase

We report a high-throughput phage selection method to identify mutants of Sfp phosphopantetheinyl transferase with altered substrate specificities from a large library of the Sfp enzyme. In this method, Sfp and its peptide substrates are co-displayed on the M13 phage surface as fusions to the phage capsid protein pIII. Phage-displayed Sfp mutants that are active with biotin-conjugated coenzyme A (CoA) analogues would covalently transfer biotin to the peptide substrates anchored on the same phage particle. Affinity selection for biotin-labeled phages would enrich Sfp mutants that recognize CoA analogues for carrier protein modification. We used this method to successfully change the substrate specificity of Sfp and identified mutant enzymes with more than 300-fold increase in catalytic efficiency with 3′-dephospho CoA as the substrate. The method we developed in this study provides a useful platform to display enzymes and their peptide substrates on the phage surface and directly couples phage selection with enzyme catalysis. We envision this method to be applied to engineering the catalytic activities of other protein posttranslational modification enzymes.     

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase,an Initial Component of the N-End Rule Pathway

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.     

Probing conformational plasticity of the activation domain of trypsin: the role of glycine hinges

Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis. The effects of these mutations on the interconversion of the zymogen-like and active conformations as well as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varying extents characterized by increased flexibility of some activation domain segments, a more accessible N-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsin transition is hindered by the mutations, which results in a shift of the equilibrium between the inactive zymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however, that the inactive conformations of the various mutants differ from each other. Binding of substrate analogues shifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsin mutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutants correlated with the proper conformation of the active site, which could be supported by varying conformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed the existence of an inactive to active conformational transition occurring prior to substrate binding.     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.

The intact, 100 kd microsomal enzyme and the 53 kd catalytic fragment of rat HMG-CoA reductase are both phosphorylated and inactivated by the AMP-activated protein kinase. Using the catalytic fragment, we have purified and sequenced peptides containing the single site of phosphorylation. Comparison with the amino acid sequence predicted from the cDNAs encoding other mammalian HMG-CoA reductases identifies this site as a serine residue close to the C-terminus (Ser872 in the human enzyme). Phosphopeptide mapping of native, 100 kd microsomal HMG-CoA reductase confirms that this C-terminal serine is the only major site phosphorylated in the intact enzyme by the AMP-activated protein kinase. The catalytic fragment of HMG-CoA reductase was also isolated from rat liver in the presence of protein phosphatase inhibitors under conditions where the enzyme is largely in the inactive form. HPLC, mass spectrometry and sequencing of the peptide containing Ser872 demonstrated that this site is highly phosphorylated in intact liver under these conditions. We have also identified by amino acid sequencing the N-terminus of the catalytic fragment, which corresponds to residue 423 of the human enzyme.     

Coenzyme B(12) dependent glutamate mutase

The crystal structure of glutamate mutase with bound coenzyme B(12) suggests a radical shuttling mechanism within the active site of the enzyme. Quantum chemical calculations of the rearrangement in combination with kinetic and mutational studies suggest the catalytic mechanism of this enzyme to proceed via a fragmentation/recombination sequence with intermediates stabilized by partial protonation/deprotonation. Crucial residues in the active site have been identified. Solution structure studies indicate the mechanism of B(12) binding to the apoenzyme.     

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.     

Effect of L-alpha-phosphatidylinositol on a vascular smooth muscle Ca2+-dependent protease. Reduction of the Ca2+ requirement for autolysis

Vascular smooth muscle contains large amounts of a Ca2+-dependent protease. Similar to a Ca2+-dependent protease previously purified from chicken gizzard smooth muscle (Hathaway, D. R., Werth, D. K., and Haeberle, J. R. (1982) J. Biol. Chem. 257, 9072-9077), the mammalian vascular muscle protease is a heterodimer consisting of 76,000- and 30,000-dalton subunits (IIa). The enzyme can undergo autolysis in the presence of Ca2+ to produce a smaller species consisting of 76,000- and 18,000-dalton subunits (IIb). Autolysis greatly reduces the Ca2+ dependence of catalytic activity. The autolytic species, IIb, was approximately 23-fold more sensitive to Ca2+ (K0.5 = 39 microM) than the native enzyme, IIa (K0.5 = 891 microM). In this communication, we report that phosphatidylinositol and to a lesser extent one metabolic derivative, dioleoylglycerol, stimulate autolysis of the vascular Ca2+-dependent protease by reducing the Ca2+ for autolysis from K0.5 = 680 microM in the absence of lipid to K0.5 = 87 microM in the presence of both phosphatidylinositol and dioleoylglycerol. Moreover, the reduction in the Ca2+ requirement for autolysis produced by the phosphatidylinositol was antagonized by the phospholipid-binding drug, trifluoperazine. In addition, the effect of phosphatidylinositol was specific for autolysis, and none of several phospholipids or derivatives tested altered the Ca2+ dependence or maximal rate for protein degradation of the autolytic product, IIb. Our results suggest that autolysis may be an important initial step in the activation of the Ca2+-dependent protease in vascular smooth muscle and that this step may be regulated by a combination of Ca2+ and phosphatidylinositol.     

Structural determinants of the half-life and cleavage site preference in the autolytic inactivation of chymotrypsin.

The molecular mechanism of the autolysis of rat alpha-chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site-directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114-->Ile and Tyr146-->His/Asn147-->Ser, that had the same enzymatic activity and molecular stability as the wild-type enzyme, displayed altered routes of autolytic degradation. The Phe114-->Ile mutant also exhibited a significantly slower autolytic inactivation (its half-life was 27-fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild-type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146-Asn147 --> Phe114 --> other sites; (b) the cleavage rates at sites Phe114 and Tyr146-Asn147 were independent from each other; and (c) the hydrolysis of the Phe114-Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half-life of chymotrypsin activity.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.