Post X‐ray crystallographic studies of chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine‐proline cluster of κ‐casein

Gustchina, Elena; Румш, Л. Д.; Ginodman, L. M.; Majer, Pavel; Andreeva, N. S.

doi:10.1016/0014-5793(95)01488-8

Cited by 29 publications

(29 citation statements)

References 17 publications

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“…Structural studies of chymosin clearly indicated that specific cleavage of the bond between Phe105 and Met106 (P1-P1′) in -casein, responsible for the high level of milk-clotting activity of this enzyme, was assisted by electrostatic interactions between a positively charged cluster His-Pro-His-Pro-His at positions P8-P4 (98–102) of the casein substrate and the negatively charged residues in loop 244–248 of chymosin (a structural equivalent of loop 238–245 in HAP)22–24. N-terminal extension beyond the P6 position of the peptide substrates also increased the catalytic efficiency of cathepsin E and of several members of the plasmepsin family25.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

Bhaumik

Xiao

Parr

et al. 2009

Journal of Molecular Biology

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The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum, as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5, 3.3, and 3.05 Å resolution, respectively. In the apoenzyme crystals HAP forms a tight dimer, not seen previously in any aspartic proteases. The interactions between the monomers affect the conformation of two flexible loops, the functionally important "flap" (residues 70-83) and its structural equivalent in the C-terminal domain (238-245), as well as the orientation of the helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active site of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer, and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different than in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared to the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.

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Section: Resultsmentioning

confidence: 99%

Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

Bhaumik

Xiao

Parr

et al. 2009

Journal of Molecular Biology

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“…The role of some of these bonds has been analyzed by site-directed mutagenesis, such as Thr77Val, and lowered catalytic efficiency was observed [66]. Interactions of pepsins with large peptides or proteins may also occur at the edge or outside the cleft, and for these cases, more substrate-binding pockets such as S 8 through S 5 subsites are anticipated [67]. Some residues, such as Lys319, far from the cleft, have been shown to be involved in catalysis by sustaining the flexibility of the enzyme [68].…”

Section: Tertiary Structurementioning

confidence: 98%

“…Bovine chymosin cleaves the Phe105-Met106 bond of k-casein of bovine milk, resulting in clotting [100]. A negatively charged region near the edge of the active-site cleft, consisting of Glu244, Asp246, Asp248, and Asp250, is involved in this specific cleavage [67], and Asp246 and Asp248 are common to all chymosins except for the chicken enzyme, being replaced by non-charged residues in pepsins A and gastricsins ( fig. 2).…”

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confidence: 97%

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

Kageyama¹

2002

CMLS, Cell. Mol. Life Sci.

229

214

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Five types of zymogens of pepsins, gastric digestive proteinases, are known: pepsinogens A, B, and F, progastricsin, and prochymosin. The amino acid and/or nucleotide sequences of more than 50 pepsinogens other than pepsinogen B have been determined to date. Phylogenetic analyses based on these sequences indicate that progastricsin diverged first followed by prochymosin, and that pepsinogens A and F are most closely related. Tertiary structures, clarified by X-ray crystallography, are commonly bilobal with a large active-site cleft between the lobes. Two aspartates in the center of the cleft, Asp32 and Asp215, function as catalytic residues, and thus pepsinogens are classified as aspartic proteinases. Conversion of pepsinogens to pepsins proceeds autocatalytically at acidic pH by two different pathways, a one-step pathway to release the intact activation segment directly, and a stepwise pathway through a pseudo-pepsin(s). The active-site cleft is large enough to accommodate at least seven residues of a substrate, thus forming S4 through S'3 subsites. Hydrophobic and aromatic amino acids are preferred at the P1 and P'1 positions. Interactions at additional subsites are important in some cases, for example with cleavage of kappa-casein by chymosin. Two potent naturally occurring inhibitors are known: pepstatin, a pentapeptide from Streptomyces, and a unique proteinous inhibitor from Ascaris. Pepsinogen genes comprise nine exons and may be multiple, especially for pepsinogen A. The latter and progastricsin predominate in adult animals, while pepsinogen F and prochymosin are the main forms in the fetus/infant. The switching of gene expression from fetal/infant to adult-type pepsinogens during postnatal development is noteworthy, being regulated by several factors, including steroid hormones.

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“…13 These features are also found in other aspartic proteases showing strong homology within the protein family. 9, [14][15][16] The substrate of chymosin, κ-casein, is a 169 residue peptide found on the surface of milk serum aggregates called casein micelles. 17 Chymosin selectively cleaves the Phe105-Met106 bond in κ-casein, which destabilises the casein micelles leading to the release of insoluble casein proteins causing milk clotting.…”

Section: Introductionmentioning

confidence: 99%

“…6,7 The HPHPH cluster is conserved in many other mammalian κcasein peptides including buffalo and goat, but in camel κ-casein the three histidine residues are mutated to arginines. 1,5 Although the allosteric activation process has been widely discussed in the literature, 1,7,16,[19][20][21] the mechanism has not been elucidated at a molecular level due to the challenges associated with studying it by experimental methods. This has hindered the development of novel enzymes and enzymatic processes for the food industry.…”

Section: Introductionmentioning

confidence: 99%

Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations

et al. 2016

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The aspartic protease, bovine chymosin, catalyzes the proteolysis of κ-casein proteins in milk. The bovine chymosin-κ-casein complex is of industrial interest as the enzyme is used extensively in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis, and crystallographic data, it has been widely reported that a HPHPH sequence in the P8-P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have employed two computational techniques, molecular dynamics and bias-exchange metadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112-116). A small conformational change in the α-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein.

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Post X‐ray crystallographic studies of chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine‐proline cluster of κ‐casein

Cited by 29 publications

References 17 publications

Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations

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