Degradation of myofibrillar proteins by trypsin-like serine proteinases.

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.     

Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).     

Benzamidine as a spectroscopic probe for the primary specificity subsite of trypsin-like serine proteinases

Summary Formation and dissociation of the benzamidine: -trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine: -trypsin complex minus the free proteinase) is 1.75 mM^–1 cm^–1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the -trypsin: BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 ± 0.5 °C. Under all the experimental conditions the -trypsin: BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine: -trypsin:BPTI adduct (7). The difference between the kinetic processes of -trypsin: BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S₁ subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.Abbreviations BPTI bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) - pNGB p-nitrophenyl-p-guanidinobenzoate - NaDodSO₄ sodium dodecyl sulfate     

Reagents for reversible coupling of proteins to the active centres of trypsin-like serine proteinases.

In order to extend the scope for the application of acyl-enzymes as fibrinolytic agents, p-amidinophenyl ester enzyme substrates were prepared that gave stabilized acyl-enzymes capable of being coupled to other proteins. Coupling was achieved by reaction of protein thiol functions with a 2-pyridyldithio moiety within the acyl group. Acyl-enzymes derived from such substrates were stable enough to permit isolation of reversible conjugates between proteins and the active centres of plasminogen activators. Hydrolytic release of active enzyme from a conjugate of human immunoglobulin G and urokinase could be demonstrated.     

Kinetic studies on the interaction of alpha 1-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases

The rates of interaction of a number of serine proteinases with a mutant form of alpha 1-proteinase inhibitor (referred to as alpha 1-proteinase inhibitor (Pittsburgh)), in which a methionine-358 to arginine-358 mutation has occurred, have been determined. An approximately 6,000-fold increase in the second order association rate constant with human thrombin was observed (48 M-1 X s-1 for the normal protein to 3.1 X 10(5) M-1 X s-1 for the arginine mutant), confirming previously observed data using bovine thrombin (Owen, M.C., Brennan, S.O., Lewis, J.H. & Carrell, R.W. (1983) New England J. Med. 309, 694-698). However, substantial increases in the rates of association with other trypsin-like enzymes were also noted, indicating that the replacement of methionine by a basic residue affects all serine proteinases with this kind of specificity. There was a marked decrease in the rates of interaction of the Pittsburgh mutant with both human neutrophil elastase and porcine pancreatic elastase, the inhibitor being converted into lower molecular mass fragments after interaction with either enzyme. Butanedione caused a substantial loss in the inhibitory activity of the arginine mutant, while having no effect on the normal protein. These data, when compared to those previously reported for differences in reaction rates between normal and oxidized alpha 1-proteinase inhibitor (Beatty, K., Bieth, J. & Travis, J. (1980) J. Biol. Chem. 255, 3931-3934), are consistent with the interpretation that the amino acid in the P1-position at the reactive site of this protein has a marked effect on determining its primary specificity.     

Degradation of collagen substrates by a trypsin-like serine protease from the fiddler crab Uca pugilator

The collagenolytic properties of a trypsin-like protease from the hepatopancreas of the fiddler crab Uca pugilator have been examined. All collagen types, I-V, were attacked by this enzyme. Types III and IV were degraded much more rapidly than types I, II, and V. Crab protease produced multiple cleavages in the triple helix of each collagen at 25 degrees C; only in the case of type III collagen, however, was a major cleavage observed at a 3/4:1/4 locus that corresponded to the region of collagen susceptibility to mammalian collagenase action. Additionally, both the affinity and the specific activity of the crab protease for native collagen were lower than those which characterize mammalian collagenase. The results of this study, in conjunction with a previous report on the collagenolytic activity of another serine protease from the fiddler crab [Welgus, H. G., Grant, G. A., Jeffrey, J. J., & Eisen, A. Z. (1982) Biochemistry 21, 5183], suggest that the following properties distinguish the action of these invertebrate collagenolytic enzymes from the metalloenzyme collagenases of mammals: (1) broad substrate specificity, including both noncollagenous proteins and collagen types I-V; (2) ability to cleave the native triple helix of collagen at multiple loci; (3) reduced affinity or higher Km for collagen; and (4) lower specific activity on collagen fibrils.     

Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins

Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in C_α-C_α distances as a criterion for loop similarity, we found that the P3-P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of C_α-C_α distances between C_α of catalytic Ser and C_α of P3-P3′ residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ₁ angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ₁ angle = −60° and −180°, respectively). To check whether the canonical conformation can be found among non–proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria—the allowed main chain dihedral angles and C_α-C_α distances for the P3-P3′ segment—were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3′ segment and with the scaffold region, P3-P4 and P3′-P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459–474, 1998. © 1998 Wiley-Liss, Inc.     

SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.     

Biotin derivatives of D-Phe-Pro-Arg-CH2Cl for active-site-specific labeling of thrombin and other serine proteinases

Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation of three biotin derivatives of the thrombin-specific inhibitor D-Phe-Pro-Arg-CH2Cl containing increasing lengths of the spacer connecting biotin. Reaction of the peptide with biotin-succinimidyl esters and purification by conventional chromatography yielded the compounds in 91-96% purity. The biotin-labeled inhibitors bound avidin with stoichiometries of 0.88-1.02 mol biotin compound/mol avidin subunits and irreversibly inactivated human thrombin with stoichiometries of 0.89-1.10 mol inhibitor/mol thrombin. Comparison of the three inhibitors by Western blotting indicated that a > or = 7- to 14-atom spacer was needed for sensitive (approximately 10 ng) detection of thrombin, with the derivative lacking a spacer only weakly detected because of its greatly reduced affinity for avidin. Application of the compounds to identify catalytically active products of factor Xa-catalyzed human prethrombin 1 activation in the absence of the protein cofactor, factor Va, allowed the direct observation of transient, low levels of the active intermediate, meizothrombin des-fragment 1, in addition to thrombin. Formation of this intermediate is concluded to reflect an intrinsic property of factor Xa activation of prethrombin 1 that is modulated by factor Va. The methods developed for preparation and characterization of the biotin-labeled inhibitors may be applicable to other tripeptide chloromethyl ketones, and the reagents can be employed for labeling of serine proteinases of diverse substrate specificity.     

Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins

Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.     

Analysis of the structure of prolactin terminal fragments as potential substrates of serine and proline-specific proteinases]

The biochemical mechanism of action of prolactin is unknown. This hormone enters the blood stream and binds to receptors predominantly in the monomeric form. A structural analysis of mammalian and piscine prolactin based on the present-day concepts of proteolytic processing of the hormone molecules in target tissues has been carried out. The experimental data suggest that prolactin molecules are protected from exopeptidase influence by their terminal cyclic peptides. The highly conservative proline-2 residues increase the resistance of the mammalian hormone N-terminal fragment to the effects of many aminopeptidases. Structurally the C-terminal cyclic peptides of prolactin, growth hormone and placental lactogen were shown to be homologous to peptides inhibiting trypsin-like proteinases. A structural analysis of the N-terminal domain of mammalian prolactin revealed the important role of Pro-2 and Pro-4 residues at positions adjacent with and inside the disulfide moiety. It is assumed that these proline residues and the cyclic structure are necessary for the manifestation of the inhibiting effect of the mammalian prolactin N-terminal dodecapeptide on proline-specific proteinases. It is assumed that proteolytic degradation of prolactin molecules in target tissues may induce the secretion of functionally active peptides.     

Thermodynamic criterion for the conformation of P1 residues of substrates and of inhibitors in complexes with serine proteinases.

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in the (Lys+)18 turkey ovomucoid third domain complex with chymotrypsin.     

A comprehensive nomenclature for serine proteases with homology to tissue kallikreins

The human kallikrein locus on chromosome 19q13.3-13.4 contains kallikrein 1--the tissue kallikrein--and 14 related serine proteases. Recent investigations into their function and evolution have indicated that the present nomenclature for these proteins is inadequate or insufficient. Here we present a new nomenclature in which proteins without proven kininogenase activity are denoted kallikrein-related peptidase. Names are also given to the unique rodent proteins that are closely related to kallikrein 1.     

New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine.

A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.     

Lack of evidence for a tetrahedral intermediate in the hydrolysis of nitroanilide substrates by serine proteinases. Subzero-temperature stopped-flow experiments

We have used a stopped-flow apparatus to reinvestigate reports, based on the observation of "burst" kinetics, of an intermediate prior to the acyl-enzyme complex in hydrolysis reactions of anilides catalyzed by trypsin and elastase [M. W. Hunkapiller, M. D. Forgac and J. H. Richards (1976) Biochemistry 15, 5581-5588; D. D. Petkov (1978) Biochim. Biophys. Acta, 523, 538-541; A. L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76, 1566-1569; P. Compton and A. L. Fink (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. We studied the hydrolysis of several anilide substrates by bovine and porcine trypsin and porcine elastase between -30 degrees C and 20 degrees C. In no case did we record true "burst" kinetics. We show that confusion spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that there is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases. The substrate N-acetyl-L-alanyl-L-prolyl-L-alanine 4-nitroanilide is a mixture of two isomers trans and cis about the L-alanyl-L-propyl peptide bond. It appears that elastase hydrolysis the cis isomer more rapidly than the trans isomer and this could lead to false "burst" kinetics. We describe the construction of the stopped-flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spectrophotometers. Solutions can be handled under anaerobic conditions. A window allows the drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range -35 degrees C to + 25 degrees C. The dead time is under 5 ms. A recording system is described that permits one to follow reactions over a wide time scale covering half-time of the order of several milliseconds to hours.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

Independent refolding of domains in the pancreatic serine proteinases

Ser-neotrypsinogen and Val-neotrypsinogen are two-chain modifications of bovine trypsinogen produced on limited proteolysis with trypsin. Ser-neotrypsinogen has Lys131-Ser132 cleaved in the connecting peptide (the autolysis loop) linking the amino- and carboxyl-terminal domains. Val-neotrypsinogen has Arg105-Val106 cleaved which is located within the amino-terminal domain. The mixed disulfide derivative of Ser-neotrypsinogen was successfully refolded. A functional molecule was regenerated from the polypeptide fragments with the correct molecular weight of neotrypsinogen in an overall yield of 7%. Val-Neotrypsinogen could not be refolded. The first-order rate constants for the regeneration of Ser-neotrypsinogen were determined from the formation of active enzyme molecules as a function of time and from the regain of the correct molecular weight. Both kinetic values were the same indicating that refolding of the polypeptide chains first forms globular domain structures. The two domains then associate and the disulfide bonds between the domains and the correct geometry of the active site residues are formed last. The same kinetic results were also found in refolding Thr-neochymotrypsinogen (Duda, C. T., and Light, A. (1982) J. Biol. Chem. 257, 9866-9871) where peptide bond cleavage also occurred in the connecting peptide. These observations support the hypothesis that the pathway of folding of serine proteinases proceeds with the independent refolding of domains.