Kazutaka Tanizawa

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar)and bovine trypsin

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.

Anionic trypsin from chum salmon: activity with p -amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.

Enzymatic coupling of α,α-dialkyl amino acids using inverse substrates as acyl donors

Abstract Two series of inverse substrates, Nα-Boc-α,α-dialkyl amino acid p-guanidino- and p-(guanidinomethyl)phenyl esters, were prepared as acyl donor components for enzymatic peptide synthesis. They were found to be readily coupled with amino acid p-nitroanilide to produce peptide. Streptomyces griseus trypsin was a more efficient catalyst than bovine trypsin.

Trypsin-catalyzed peptide synthesis withm-guanidinophenyl andm-(guanidinomethyl)phenyl esters as acyl donor component

SummaryTwo series of inverse substrates,m-guanidinophenyl andm-(guanidinomethyl)phenyl esters derived fromN-(tert-butyloxycarbonyl)amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such asl-alaninep-nitroanilide to produce dipeptide in the presence of trypsin.Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.

Biologically selective potassium channel openers having 1,1-diethylpropyl group

To find out selective potassium channel openers (PCOs), we synthesized several 3,5-disubstituted phenylcyanoguanidine derivatives and investigated their structure-activity relationships (SAR). As a result, we discovered selective PCOs having 1,1-diethylpropyl group toward antihypertensive activity.

Chum salmon trypsin-catalyzed peptide synthesis with inverse substrates as acyl donor components at low temperature

Abstract Chum salmon trypsin-catalyzed peptide synthesis has been studied by using p -amidinophenyl esters of N α -( tert -butyloxycarbonyl)amino acid as the acyl donor components at 0°C. The reaction conditions were optimized for an organic solvent, pH, and concentration of the acyl acceptor. The method was shown to be successful as a general method for the synthesis of the peptide, and also useful for the preparation of peptides containing d -amino acids. The enzymatic hydrolysis of the resulting products was negligible.