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.

Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases.

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely, although not proven, that these propeptides are functionally important in the active enzymes, as well as in the zymogens. A mutant chymotrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., et al. (1996) FEBS Lett. 379, 139-142]. In this paper, we investigate the role of the disulfide-linked propeptide in the conformational stability of chymotrypsin(ogen). We compare the stabilities of the wild-type and mutant proteins (lacking propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) and active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experiments, we determined that covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (DeltaDeltaG). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin. This may also explain (besides keeping the zymogen inactive) the evolutionary conservation of the propeptide-enzyme interactions. Tryptophan fluorescence, circular dichroism, microcalorimetric, and activity measurements suggest that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appears to be accomplished via alternative interdomain contacts.

The differential specificity of chymotrypsin A and B is determined by amino acid 226.

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydrophobic residues, like tryptophan, phenylalanine and tyrosine. We found, however, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phenylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all these substrates with comparable catalytic efficiency. Analysing the structure of substrate binding pocket of chymotrypsin A prompted us to perform an Ala226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsible for the differential specificities of chymotrypsin A and B isoenzymes.

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin --> trypsin specificity conversion.

Expression of rat chymotrypsinogen in yeast: a study on the structural and functional significance of the chymotrypsinogen propeptide.

The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild-type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1-Cys122 disulfide-linked propeptide show that this linkage is not important either for activity or substrate specificity. However this linkage appears to be the major factor in keeping the zymogen stable against non-specific activation. A comparison of zymogen stabilities showed that the trypsinogen propeptide is ten times more effective than the chymotrypsinogen propeptide in preventing non-specific zymogen activation during heterologous expression and secretion from yeast. This feature can also be transferred in trans to chymotrypsinogen; i.e. the chymotrypsin trypsin propeptide chimera forms a stable zymogen.

Attempts to convert chymotrypsin to trypsin.

Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin-->trypsin specificity conversion.

A rapid and effective procedure for screening protease mutants.

We describe a simple and effective procedure to screen for active proteases among a large number of mutants. First, the mutants are genetically tested by the protease activity produced in the periplasm of transformed bacteria which supplies the cells with a nitrogen source by hydrolyzing a protein applied to plates. Then a less sensitive activity staining and an X-ray film digestion assay are used to verify and estimate the activity of the mutants that proved to be positive in the first step. Depending essentially on the level of periplasmic protease activity, the method can detect both the activity and the stability of the expressed enzymes. We calibrated the method with transformants that produce wild-type trypsin, chymotrypsin and trypsin mutants of known activity. Using this method we found two active revertants of the inactive Asn102 trypsin mutant, by screening approximately 4.4 x 10(4) random mutants that were generated by the polymerase chain reaction on a cDNA fragment. This procedure should be useful in searching for proteases of novel specificity and/or reaction chemistry engineered by random mutagenesis, and also for in vitro evolution studies.

Different cholesterol fractions, LCAT activity and lipid peroxides in the serum of children whose parents had early coronary heart disease.

Children whose parents had early coronary heart disease were investigated for lipid abnormalities. In order to assess high risk parameters and the efficacy of the applied care, serum total cholesterol (TC), total triglyceride (TT), total free cholesterol (FC) high density lipoprotein cholesterol (HDLC), high density lipoprotein free cholesterol (HDLFC), lipid peroxide (LP) levels, and lecithin: cholesterol acyl transferase (LCAT) activity were measured. Compared to a group of control children, the offspring of high risk subjects had increased TC, FC, and LP levels and decreased HDLC levels. After one year of preventative care all parameters normalized except the high FC level and elevated LCAT activity. The measurement of serum FC and LP levels seems to be a useful method for the determination of true high risk. The LCAT activity may show the efficacy of the dietary treatment.

Serum lipids, lipid peroxides and the care of children with high risk atherosclerotic family history.

Children of parents who had their first acute myocardial infarction (AMI) before the age of 45 years were investigated and divided into 2 groups. Group 1 consisted of 12 children: both their parents or one parent and grandparents suffered from AMI. Group 2 consisted of 55 children, each of whom had one parent with AMI. Serum levels of total cholesterol (TC) and lipid peroxide (LP) were higher in these children and HDL-cholesterol values were lower than those of 39 control children. The extent of serum TC and HDL-C differences was similar in both high risk groups but the serum LP level was significantly higher in group 1 than in group 2. The serum free cholesterol (FC) level was higher in group 1 than in controls. Children were treated according to a program giving advice on diet, physical exercise and health education for 1 year. At the end of this period the serum HDL-C level increased in both groups, while LP and TC levels decreased. The high serum FC level did not change.

Kinetics of NADPH-dependent lipid peroxidation and a possible initiation-preventing antioxidant effect of microsomal (+)-alpha-tocopherol.

The initial part of NADPH-driven lipid peroxidation was investigated in liver microsomes. Without the addition of any antioxidant or pretreatment of animals with vitamin E, a delay was observed in the malondialdehyde and lipid hydroperoxide formation, but not in oxygen consumption. The duration of lag and the effect of ADP-Fe2+ on it showed differences in rat, mouse, chicken and rabbit microsomes. As it was not caused by cytoplasmic contaminations, this lag was an indication of the antioxidant capacity of microsomes, possibly due to the oxidation of their (+)-alpha-tocopherol content. The length of lag was dependent on the NADPH-cytochrome-P-450 reductase activities and the concentration of Fe-ion complexes. The results presented here suggest that (+)-alpha-tocopherol acted during the lag as an initiation-preventing rather than a chain-breaking antioxidant in rat liver microsomes. The lag may explain the known differences found in the inducibility and intensity of lipid peroxidation of microsomes from various species, and provides means to elucidate the molecular mechanism of vitamin E action against free radicals formed in a membrane of biological origin.

Effect of a heme-peptide derived from cytochrome-c on lipid peroxidation. II. Experiments with liver microsomes.

ADP-Fe2+ stimulated, NADPH dependent lipid peroxidation of liver microsomes (as measured by malondialdehyde formation) was not inhibited by c-heme-nonapeptide, unlike the same process in brain microsomes. However, in the presence of 5 mM aminopyrine (causing partial inhibition) or SKF-525A (a specific inhibitor of cytochrome P450) the residual activity of lipid peroxidation of liver microsomes was markedly inhibited by c-heme-nonapeptide. Further, c-heme-nonapeptide itself prevented the transient accumulation of lipid hydroperoxides during ADP-Fe2+ stimulated lipid peroxidation. These results led us to suggest two different pathways of lipid peroxidation. The first route involves cytochrome P450. The second pathway, which can be inhibited by c-heme-nonapeptide, appears to be more important physiologically.

Inhibition of lipid peroxidation by heme-nonapeptide derived from cytochrome c.

Heme-nonapeptide, derived from cytochrome c, inhibited both the NADPH- and NADH-dependent lipid peroxidation of brain microsomes but, in the case of liver microsomes, this inhibitory effect manifested itself in the presence of SKF-525A (a specific blocker of cytochrome P-450) only. Heme-nonapeptide prevented the transient accumulation of lipid peroxides in microsomes during lipid peroxidation. The oxygen consumption of microsomes in the presence of NADPH or NADH was stimulated by heme-nonapeptide. From these results we concluded that, in vitro, there are two independent mechanisms of lipid peroxidation in liver microsomes. It is suggested that, in vivo, the heme-peptide-sensitive mechanism, observed in brain microsomes, is more important.