Covalent structure of bovine chymotrypsinogen A

Meloun, B.; Kluh, I.; Kostka, V.; Morávek, L.; Prusík, Z.; Vanĕcek, J; Keil, B.; Šorm, F.

doi:10.1016/0304-4165(66)90258-3

Cited by 63 publications

(20 citation statements)

References 21 publications

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“…The sequence of aCT was obtained from ref. 29 and the x-ray coordinates are known to 1.68-A resolution (30). The bovine trypsin sequence and x-ray coordinates were from refs.…”

Section: Discussionmentioning

confidence: 99%

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Atassi

Manshouri

1993

Proc. Natl. Acad. Sci. U.S.A.

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Two 29-residue peptides were prepared, one of which (ChPepz) was designed by surface-simulation synthesis to mimic the active site of a-chymotrypsin, and the other (TrPepz), which contained four substitutions relative to ChPepz, was fashioned after the active site of trypsin. Each peptide was cyclized by a disulfide bond. The ChPepz monomer effected hydrolysis of the ester group in N-benzoyl-L-tyrosine ethyl ester, an a-chymotrypsin substrate, with Km and kt values that were comparable to those of a-chymotrypsin. ChPepz was completely inactivated by diisopropyl fluorophosphate (DIFP), L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK), or reduction of the disulfide bond. It had no catalytic activity on N-tosyl-L-arginine methyl ester, a trypsin substrate. On the other hand, TrPepz, which had no effect on N-benzoyl-L-tyrosine ethyl ester, hydrolyzed N-tosyl-L-arginine methyl ester with a Km value that was essentially identical to that of trypsin, but its k5,,, value was almost half that of trypsin. TrPepz was fully inactivated by reduction of the disulfide bond, by DIFP, or by phenylmethylsulfonyl fluoride but not by TPCK. It was also completely inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and human a1-antitrypsin. ChPepz and TrPepz hydrolyzed proteins (myoglobin and casein) to give panels ofpeptides that were similar to those of the same protein obtained with the respective enzyme. However, TrPepz was more efficient than trypsin at hydrolyzing the C bonds of two or more consecutive lysine and/or arginine residues. Like its esterase activity, the proteolytic activity of ChPepz was inhibited by either DIFP or TPCK whereas that of TrPepz was inhibited by either DIFP or phenylmethylsulfonyl fluoride but not by TPCK. Finally, ChPepz and TrPepz were each more active at low temperature than the respective enzyme. This ability to construct fully functional peptide enzymes (pepzymes) of chosen specificities should find many practical applications.The critical role of enzymes in the catalysis of biological processes has, for decades, made enzymes the subject of the most intense studies. The duplication of the activities of enzymes by synthetic analogs has been a prime goal of biochemists. X-ray crystallography of enzymes and their complexes with substrate analogs or inhibitors has provided detailed information about the architecture and contact residues ofthe active sites of many enzymes. In 1976, Atassi and coworkers (1, 2) devised the technique of "surfacesimulation" synthesis, in which the spatially adjacent residues constituting a protein binding site are directly linked via peptide bonds with appropriate spacing and directionality. A peptide is thus generated that does not exist in the protein but mimics the conformation and disposition of the residues of the binding site. Surface-simulation synthesis (for review, see ref.3) has been employed to mimic protein antigenic sites (1, The publication costs of this article were defrayed in part by page charge payment. This arti...

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“…The sequence of aCT was obtained from ref. 29 and the x-ray coordinates are known to 1.68-A resolution (30). The bovine trypsin sequence and x-ray coordinates were from refs.…”

Section: Discussionmentioning

confidence: 99%

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Atassi

Manshouri

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…With respect to the N-terminal sequences of the heavy chains of acrosin and several other serine proteinases, Hedrick et al [31] found a positional identity ranging between 33% and 48%. If one compares the complete amino acid sequence of the boar acrosin heavy chain, excluding the proline-rich C-terminal domain (AIa275 -Leu385) with those of other serine proteinases, positional identities of 36.6% with bovine trypsinogen [32], 32.7% with bovine chymotrypsinogen A [33], 30% with bovine chymotrypsinogen B [34], 32.8% with trypsin I of the crayfish [35], 30.2% with the human protein-C precursor [36] and 36.6% with the human plasminogen [37] can be determined.…”

Section: Analysis Of the Amino Acid Sequence Of Hoar Preproacrosin Dementioning

confidence: 99%

Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis

Adham

Klemm

Maier

et al. 1989

European Journal of Biochemistry

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Complementary DNA clones for the boar preproacrosin have been isolated from a randomly primed testis cDNA library in LgtlO and from an oligo(dT)-primed testis cDNA in Agtll. The nucleotide sequence of the 1418-bp cDNA insert includes a 46-bp 5'-untranslated region, an open reading frame of 1248 bp corresponding to 416 amino acids (45.59 kDa) and a 121-bp 3'-untranslated region. The deduced amino acid sequence includes the active-site residues histidine, asparagine and serine of the catalytic triad of the serine proteinase super-family and is colinear with that determined by amino acid sequencing of the boar acrosin light chain and of a small region of the NH2-terminal sequence of the heavy chain. The preproacrosin cDNA contains at the 3' end a 381-bp sequence which codes for an amino acid sequence not yet found in any other serine proteinase. This amino acid sequence is rich in proline (42 out of 127 amino acids) and is suggested to be involved in the recognition and binding of the spermatozoa to the zona pellucida of the ovum. The mRNA for preproacrosin is synthesized as an approximately 1.6-kb-long molecule only in the postmeiotic stages of boar and bull spermatogenesis.Mammalian fertilization is the result of a poorly understood complex series of biochemical events that occur when an ovum and a spermatozoa fuse. Acrosin, a serine proteinase located in the acrosome of the sperm, has been implicated in the recognition and binding of the sperm to the zona pellucida of the ovum [l -31 and the sperm penetration through the zona pellucida [4, 51. The enzyme is synthesized in a zymogen form, proacrosin, which is activated to the mature enzyme during the so-called acrosome reaction [6]. Immunofluorescent studies indicate that the biosynthesis of proacrosin starts in early haploid spermatids [7, 81. Despite the great importance of proacrosin/acrosin in the fertilization process, only very few data as to its molecular structure are available, mostly in the boar. In this species, the single-chain glycoprotein of proacrosin has an estimated molecular mass of 55 kDa [6]. During its activation, the twochain glycoprotein a-acrosin is formed with a light chain and a heavy chain held together by two disulfide bridges [9]. The 4.2-kDa light chain consists of 23 amino acid residues and contains one carbohydrate attachment site. The 37-kDa heavy chain with approximately 320 amino acids was found to con-

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“…As well as trypsin, this enzyme is found in the pancreas of various animals and it is the main contaminant of trypsin. By itself, this enzyme corresponds to an equimolar mixture of chymotrypsin A (Hartley, 1964;Meloun et al, 1966) and chymotrypsin B (Smillie, Furka, Nagabhushan, Stevenson, & Parkes, 1968) with 80% of identity between both sequences but with the two forms having similar activity (Gráf, Szilágyi, & Venekei, 1998).…”

Section: Chymotrypsinmentioning

confidence: 99%

Acceleration and Improvement of Protein Identification by Mass Spectrometry

Bienvenut¹

2005

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Covalent structure of bovine chymotrypsinogen A

Cited by 63 publications

References 21 publications

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis

Acceleration and Improvement of Protein Identification by Mass Spectrometry

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