Trypsin catalytic activity

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

In addition, it has been shown that other enzymes such as trypsin can be successfully immobilized and used for the conversion of substrates with higher molecular masses [76]. Petro et al. [94] compared the activity of trypsin immobilized on macroporous beads and on monolithic supports. They were able to show that the catalytic activity of trypsin bound to a monolith was much higher and resulted in a much higher throughput. Other enzymes such as invertase [76] and... 

S ATP -I- myosin I heavy chain <1, 2, 9-11> (<10> major site of phosphorylation is Ser8 [22] <2> 35 kDa trypsin fragment of the C-terminus of the maximally activated, phosphorylated enzyme is fully catalytically active and contains 2 thirds of the autophosphorylation sites of the native enzyme [20] <9,10> substrate myosin ID [19,22] <2> higher activity with membrane-bound substrate myosin I [17] <2> substrates are heavy chains of myosin lA and IB [6,7,17] <2,11> substrate is heavy chain of myosin IC [7,23,24] <2> a basic amino acid is essential on amino-terminal side of phosphorylation site, two are preferable, and a Tyr-residue is essential two residues away on the COOH-terminal side [7] <2> contains two myosin heavy chain kinases one for myosin I and one for myosin II... 

For example, if either the histidine or the serine of the triad of subtilisin was replaced by alanine the catalytic activity decreased by a factor of 2 x 106 and replacement of the aspartate of the triad by alanine decreased activity by a factor of 3 x 104.229/258 When Asp 102 of trypsin is replaced by asparagine the catalytic activity falls by four orders of magnitude.259 This may be in part because the histidine in this mutant is hydrogen bonded to Asn 102 as the tautomer with a proton on Ne, the nitrogen that should serve as the catalytic base in step b (Fig. 12-11).260 A mutant in which Ser 214 (see Fig. 12-10) was replaced with alanine is fully active but charged residues in this position interfere with catalysis.261... 

Trypsin in which Asp 102 has been replaced by Asn has 1 ()4 times less catalytic activity than natural trypsin at neutral pH. From the crystal structure of the mutant enzyme it appears that the imidazole group of His 57 is held by the Asn side chain in the wrong tautomeric form for catalysis. Explain. Compare this incorrect tautomeric form with that in the initial structure shown in Fig. 12-11. 

A different type of covalent regulation of enzyme activity is the enzyme-catalysed activation of inactive precursors of enzymes (zymogens) to give catalytically active forms. The best examples are the digestive enzymes, e.g. trypsin. Proteolytic enzymes would digest the inside of the cells that produce the enzyme, so they are produced in an inactive form which is activated to the true enzyme once they have entered the digestive system of the animal. 

Schematic diagrams of the amino acid sequences of chymotrypsin, trypsin, and elastase. Each circle represents one amino acid. Amino acid residues that are identical in all three proteins are in solid color. The three proteins are of different lengths but have been aligned to maximize the correspondence of the amino acid sequences. All of the sequences are numbered according to the sequence in chymotrypsin. Long connections between nonadjacent residues represent disulfide bonds. Locations of the catalytically important histidine, aspartate, and serine residues are marked. The links that are cleaved to transform the inactive zymogens to the active enzymes are indicated by parenthesis marks. After chymotrypsinogen is cut between residues 15 and 16 by trypsin and is thus transformed into an active protease, it proceeds to digest itself at the additional sites that are indicated these secondary cuts have only minor effects on the enzymes s catalytic activity. (Illustration copyright by Irving Geis. Reprinted by permission.)...

The pancreatic enzyme it stored and secreted as a proenzyme with an additional seven residues at the N-tcmiinus, The proenzyme serves, like the other pancreatic proteinase zymogens, to prevent aotodigestion of the pancreatic cells. Upon secretion into the gastrointestinal tract, trypsin cleaves off these seven residues to produce the enzymatic form with full activity on insoluble substrates. On monomeric substrates, however, there is little difference between the catalytic activity of the proenzyme and of the activated enzyme [34],... 

The /8 subunit of tryptophan synthase from E. coli is inactivated by a mutation at Gly-281 (G281R).108 Gly-281 is located at a sharp turn in the trypsin-sensitive, subdomain (residues 260-310) that makes several contacts with the a subunit.7) The G281R mutation alters the catalytic properties of the isolated /8 subunit and weakens association with the a subunit. The mutation may interfere with hydrophobic interactions between the N-terminal and C-terminal domains of the /8 subunit and prevent a conformational change that affects catalytic properties and subunit interaction.108 Insertion of arginine or tryptophan between tyrosine 279 and phenylalanine 280 of the /3 subunit greatly weakens subunit interaction and decreases catalytic activity (X.-J. Yang and E.W. Miles, unpublished results). 

Fig. 5. Productive [a), c)] and non-productive [b), d) f)] binding of tyrosine derivatives to chymo-trypsin. Catalytic residues in the active site are illustrated as a sharp edge...

Photostimulation of the catalytic activities of bio-imprinted enzymes was studied by Willner and co-workers [25]. The goal of this study was to control the enzymatic activity with light. They linked a photoactive group such as nitropyran to a-chymo-trypsin. The resulting conjugated enzyme was allowed to interact with A -acetyl-L-phenylalanine (as template) in an an aqueous medium. After precipitating the enzyme-template complex, the template was carefully eluted from the enzyme. The catalytic activities of the non-imprinted and imprinted photoactive enzymes were... 

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