The nature of the rennin — Sensitive bond in casein and its possible relation to sensitive bonds in other proteins

Hill, R. D.

doi:10.1016/0006-291x(68)90346-x

Cited by 37 publications

(14 citation statements)

References 12 publications

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“…The S8–S4 pockets have previously been shown to be important for binding. It has been demonstrated that the rate of hydrolysis is ∼18-fold higher for the fragment P8 – P4 ′ compared to the P3 - P4 ′ fragment. , Experimentally, it is known that the dipeptide H-Phe-Met-OH is not easily hydrolyzed by bovine chymosin and neither are tri- or tetrapeptides containing the scissile bond . Hill stated, on the basis of a series of experiments, that other residues were needed to modulate the binding and subsequent cleavage.…”

Section: Resultsmentioning

confidence: 99%

Hot-Spot Mapping of the Interactions between Chymosin and Bovine κ-Casein

Sørensen

Palmer²,

Schiøtt

2013

J. Agric. Food Chem.

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Chymosin is a commercially important enzyme in the manufacturing of cheese. Chymosin cleaves the milk protein κ-casein, which initiates the clotting process. Recently, it has been shown that camel chymosin has superior enzymatic properties toward cow's milk, compared to bovine chymosin. The two enzymes possess a high degree of homology. There are only minor differences in the binding cleft; hence, these must be important for binding the substrate. Models for the binding of a 16 amino acid fragment, consisting of the chymosin-sensitive region of bovine κ-casein (97-112), to both enzymes have previously been presented. Computational alanine scanning for mutating 39 residues in the substrate and the bovine enzyme are presented herein, and warm- (ΔΔG > 1 kcal/mol) and hot-spot (ΔΔG > 2 kcal/mol) residues in the bovine enzyme are identified. These residues are relevant for site-directed mutagenesis, with the aim of modifying the binding affinity and in turn affecting the catalytic efficacy of the enzyme.

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Section: Resultsmentioning

confidence: 99%

Hot-Spot Mapping of the Interactions between Chymosin and Bovine κ-Casein

Sørensen

Palmer²,

Schiøtt

2013

J. Agric. Food Chem.

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“…This finding suggested a high specificity for Phe-Met bonds, but the peptide methyl esters Phe-Met-OMe, Phe-Met-AlaOMe, and Leu-Phe-Met-Ala-OMe were hydrolyzed only to a negligible extent (70). These results showed that the mere existence of the dipeptide, even with the end-groups blocked, was not enough for hydrolysis to take place.…”

Section: Chymosin (Rennin)mentioning

confidence: 93%

Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Svendsen

1976

Carlsberg Res. Commun.

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Subtilisin type Carlsberg and subtilisin BPN' are two well characterized extracellular proteases from Bacillus subtilis and Bacillus amyloquefaciens, respectively. The present review summarizes the various chemical modification studies which have been performed with these enzymes. Of special interest has been those modifications which lead to changes in the enzymatic properties with regard to the hydrolysis of polypeptide substrates but not small synthetic ester substrates. In this way it is possible to obtain information about how large an area of the surface of the enzyme is involved in the binding of natural substrates (e.g. clupein, gelatine, casein). Nitration, iodination, glutarylation or succinylation of the subtilisins leads to increases in the hydrolyses of clupein and gelatine, while modification of carboxyl groups leads to a decrease. In all these cases the hydrolysis of small ester substrates is almost unaffected. A section of the review deals with the comparison between the results obtained by chemical modification studies and the two other methods available for the study of "secondary binding sites": X-ray diffraction analyses and kinetic studies with synthetic polypeptides. The productive binding mode for polypeptides proposed by KRAUT and coworkers is in agreement with the results obtained by nitration of the subtilisins. However, results from our laboratory and the literature point towards more than one mode of productive binding. These results are discussed. Finally a comparison is made between the secondary binding sites in subtilisin and other proteolytic enzymes, especially the serine proteases. The importance of secondary interactions between enzyme and substrate is discussed.

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“…Both the length of the peptide and the sequence around the sectile bond are important determinants of enzymesubstrate interaction. Serine 104 appears to be particularly important (Hill, 1968(Hill, , 1969 and its replacement by Ala in the above pentapeptide renders the Phe-Met bond very resistant to hydrolysis by chymosin (Raymond et aI., 1972) but not by pepsins (Raymond and Bricas, 1979); even substituting D-Ser for L-Ser markedly reduced its sensitivity (Raymond and Bricas, 1979). Extension of the above pentapeptide from the N-and/or C-terminal to reproduce the sequence of K-casein around the chymosin-susceptible bond increases the efficiency with which the Phe-Met bond is hydrolysed by chymosin (Visser et at., 1976(Visser et at., , 1977 (Table 1.1).…”

Section: Primary Phase Of Rennet Actionmentioning

confidence: 96%

“…4.7 (Hill, 1968;de Koning et al, 1978;Raymond and Bricas, 1979) but is 5.3-5.5 on K-CN f His9S-LYSlll (Visser et al, 1987). Chymosin hydrolyses insulin, acid-denatured haemoglobin and Na-caseinate optimally at pH 4.0, 3.5 and ca.…”

Section: Phmentioning

confidence: 97%

“…The dipeptide, H.Phe-Met.OH, is not hydrolysed, nor are tri-or tetrapeptides containing a Phe-Met bond. However, this bond is hydrolysed in the pentapeptide, H.Ser-Leu-Phe-Met-Ala-OMe (Hill, 1968(Hill, , 1969 and reversing the pbsitions of serine and leucine, to give the correct sequence of K-casein, increases the susceptibility of the Phe-Met bond to chymosin (Schattenkerk and Kerling, 1973). Both the length of the peptide and the sequence around the sectile bond are important determinants of enzymesubstrate interaction.…”

Section: Primary Phase Of Rennet Actionmentioning

confidence: 97%

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