Pepsin crystallization

Early protein crystallographers, proceeding by analogy with studies of other crystalline substances, examined dried protein crystals and obtained no diffraction patterns. Thus X-ray diffraction did not appear to be a promising tool for analyzing proteins. In 1934, J. D. Bernal and Dorothy Crowfoot (later Hodgkin) measured diffraction from pepsin crystals still in the mother liquor. Bernal and Crowfoot recorded sharp diffraction patterns, with reflections out to distances in reciprocal space that correspond in real space to the distances between atoms. The announcement of their success was, in effect, a birth announcement for protein crystallography. 

Bernal and Crowfoot first succeeded in finding the reason for these failures. They showed with pepsin crystals that genuine X-ray diffraction patterns can be obtained when the crystals are investigated while enclosed in their own mother liquor (in the drying of the crystals customary up till then a profound change in internal structure — denaturation — can occur). 

The results above indicated that the mutations had a minimal effect on substrate binding and influenced primarily the properties of enzyme-bound species in the reaction pathway. Additionally, stereochemical analysis of peptide bond hydrolysis (James and Sielecki, 1985) and pepsin crystal stmcture (Sielecki et al., 1990) indicated that glycine 76 is in a position most favorable for interactions with reaction intermediates. A possible involvement of glycine 76 in stabilizing the transition state was suggested as a h5T)othetical catalytic mechanism for pepsin (Pearl, 1987). As indicated above, all the mutants had altered kinetic constants compared to... 

The pepsin crystals used in the x-ray crystallographic studies are prepared at the presence of ethanol (1,2). It is quite possible that ethanol molecules can eventually be identified on the three-dimensional structure of pepsin crystals to confirm the kinetic studies. 

From these data it was concluded that pepsin has an extended active site being able to accomodate specifically five amino acid residues of the substrate. The orientation of the substrate molecule relative to the ethanol binding loci in pepsin crystals has been determined. Pepsin mechanism includes "amino-enzyme" formation which chemically is not an amide, formed by the enzyme carboxyl with the amino fragment of the substrate. 

The second enzyme to be crystallized (byjohn Nordrrnp in 1930). Even more than nrease before it, pepsin. study by Northrnp established tirat enzyme activity comes from proteins. fAiso known as rennin, it is tire major pepsinlike enzyine in gastric Jnice of fetal and newborn animals. 

There are indications that the crystal protein is subject to proteolytic enzymes when separated from the sporangium. The crystal protein has also been shown to be degradable by fairly nonspecific proteases such as pepsin and trypsin. 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

The mutation of the hydroxyl group positioned in R-configuration at the C(3) atom of the central statine (rSta) residue of the inhibitor gives rise to AAGbind of -0.51 kcal/mol, which is very close to the experimental value of -0.8 kcal/mol. It may be noted here that the starting configuration of the inhibitor in the enzyme-inhibitor complex is the same as that of pepstatin. The crystal structure of rhizopus pepsin or any other aspartic proteinase... 

Globular proteins were much more difficult to prepare in an ordered form. In 1934, Bernal and Crowfoot (Hodgkin) found, that crystals were better preserved if they were kept in contact with their mother liquor sealed in thin-walled glass capillaries. By the early 1940s crystal classes and unit cell dimensions had been determined for insulin, horse haemoglobin, RNAase, pepsin, and chymotrypsin. Complete resolution of the structures required identification of the crystal axes and some knowledge of the amino acid sequence of the protein—requirements which could not be met until the 1950s. 

Finds crystal structure of peptides bound to Rhizopus pepsin... 

The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early enzyme studies. Sumner found that urease crystals consisted entirely of protein, and he postulated that all enzymes are proteins. In the absence of other examples, this idea remained controversial for some time. Only in the 1930s was Sumner s conclusion widely accepted, after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins. During this period,... 

Pepsin is secreted as the inactive pepsinogen, which is activated by H+ ions at a pH below 5. Determination of its crystal structure revealed that in the proenzyme the N-terminal 44-residue peptide segment lies across the active site, blocking it.384 At low pH the salt bridges that stabilize the proenzyme are disrupted and the active site is opened up to substrates. 

There are four basic mechanistic classes of enzyme which catalyse the hydrolysis of peptide bonds serine proteinases such as trypsin and chymo-trypsin, cysteine proteinases such as papain, acid (aspartic) proteinases such as pepsin, and zinc-containing metalloproteinases such as carboxypeptidase. X-ray crystal structures of representative examples of each class of enzyme are available, and the detailed reaction pathways probably taken by all four classes of enzyme have been subject to analysis in terms of ALPH, These analyses have been for the most part permissive rather than compelling, and are considered in turn below. 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

On starch gel electrophoresis, gastricsin migrated 6.5 cm toward the anode after 22 hours as one single narrow band in acetate buffer of pH 5.0. These two materials also differed in heat sensitivity while pepsin at pH 2.0 and 65° C lost 69% of its activity, gastricsin under these conditions lost 44.8%. Conversely, at pH 3.2 pepsin was inactivated only 11.2%, while gastricsin was inactivated 22.3% (Fig. 4). Both enzymes hydrolyzed synthetic carbobenzoxy-glutamyl-L-tyrosine, which is a specific substrate for pepsin. Gastricsin was crystallized as it came from... 

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