Hans Neurath

Evolution of structure and function of proteases.

One of the striking features of the proteolytic enzymes as a group is the immense variety of biological functions served by enzymes employing one of a few basic mechanisms. For example, in the higher animals, enzymes for activation of zymogens (trypsin), for digestion of dietary proteins (trypsin, chymotrypsin, elastase), for blood clotting (thrombin), for clot lysis (plasmin), and for sensing pain (kallikrein) all appear to use the same mechanism and to have evolved from the same ancestral gene by the process of gene duplication and subsequent divergent evolution. Equally striking is the variety of chemical solutions of the same functional problem, such as the peptide-bond cleavage by sulfhydryl proteases on the one hand and serine proteases on the other.

Proteolytic processing and physiological regulation

Proteolytic processing is a common and effective mechanism of physiological regulation. The basic principle is a conformational change induced in the protein precursor by the post-translational proteolytic cleavage of a specific peptide bond. The extension of earlier studies of model zymogens to more complex systems of physiological regulation, using methods of both protein chemistry and molecular biology, has enormously extended knowledge of the repertoire of proteolytic processing reactions and has contributed significantly to current studies of the structure, domain organization and evolution of proteins.

The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships

Abstract The sequence of all presently known trypsin-related serine proteases and their zymogens of animal and bacterial origin were optimally aligned on the basis of three different scoring schemes for amino acid comparisons. Sequence homology was found to extend into the activation peptides. The gaps resulting from the alignment of the sequences of the active enzymes formed the basis for a new procedure based on position and number of gaps, which allowed the correct topology of the evolutionary relationship of thrombin and the pancreatic enzymes trypsin, chymotrypsin and elastase to be determined. The procedure was applied in an analogous manner to changes in disulfide bridges as well as to a selected set of amino acid positions. Evolutionary distances between proteins were estimated by minimum, base differences as well as according to the stochastic model of evolution . These distances were used successfully to find the best topology of evolutionary relationships. The fact that the branch lengths in evolutionary trees were less affected by the number of sequences considered when evolutionary distances between contemporary sequences were measured in minimum base differences than when measured according to the stochastic model of evolution, suggested in our specific case, that minimum base differences yielded estimates of evolutionary distance closer to reality than the stochastic model of evolution. All these techniques combined yielded the following picture for the evolution of the four protease families. Prothrombin and the zymogens of the pancreatic serine proteases had a common ancestor with tryptic specificity. After the initial divergence, the gene for trypsinogen duplicated. Evidence was found that the duplicated gene underwent drastic changes for a short period of time to become eventually the common ancestor of chymotrypsin and elastase. The phylogenetic tree elaborated for these enzyme families and the methods introduced to determine its topology, should readily allow determination of the attachment site of branches leading to newly sequenced serine proteases, provided their amino acid sequence can be aligned fairly unambiguously. In addition, the consequences of the alignment of the different serine proteases for the relationship of zymogen to enzyme are discussed.

The structure of rat mast cell protease II at 1.9-A resolution.

The structure of rat mast cell protease II (RMCP II), a serine protease with chymotrypsin-like primary specificity, has been determined to a nominal resolution of 1.9 A by single isomorphous replacement, molecular replacement, and restrained crystallographic refinement to a final R-factor of 0.191. There are two independent molecules of RMCP II in the asymmetric unit of the crystal. The rms deviation from ideal bond lengths is 0.016 A and from ideal bond angles is 2.7 degrees. The overall structure of RMCP II is extremely similar to that of chymotrypsin, but the largest differences between the two structures are clustered around the active-site region in a manner which suggests that the unusual substrate specificity of RMCP II is due to these changes. Unlike chymotrypsin, RMCP II has a deep cleft around the active site. An insertion of three residues between residues 35 and 41 of chymotrypsin, combined with concerted changes in sequence and a deletion near residue 61, allows residues 35-41 of RMCP II to adopt a conformation not seen in any other serine protease. Additionally, the loss of the disulfide bridge between residues 191 and 220 of chymotrypsin leads to the formation of an additional substrate binding pocket that we propose to interact with the P3 side chain of bound substrate. RMCP II is a member of a homologous subclass of serine proteases that are expressed by mast cells, neutrophils, lymphocytes, and cytotoxic T-cells. Thus, the structure of RMCP II forms a basis for an explanation of the unusual properties of other members of this class.

The molecular weight of a-chymotrypsinogen

The molecular weight of crystalline a-chymotrypsinogen has been determined from amino acid analysis, light-scattering, and sedimentation-diffusion measurements. The values obtained by the use of these methods are, respectively, 25,100, 26,000 and 24,200. These values, together with the recently reported X-ray estimate of 25,000, converge toward 25,000 as the most probable molecular weight of a-chymotrypsinogen.

From proteases to proteomics

This personal and professional autobiography covers the 50‐yr period of 1950–2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

Structural changes in the activation of chymotrypsinogen and trypsinogen. Effect of urea on chymotrypsinogen and delta-chymotrypsin

Abstract In an attempt to detect structural differences between chymotrypsinogen and chymotrypsin, and trypsinogen and trypsin, measurements of the optical rotation and of the enzymatic activity of these proteins were carried out. The results indicate that the decrease in specific levorotation on activation of these zymogens is correlated with the appearance of enzymatic activity, and that the structure of the active enzyme is more sensitive to pH changes than that of the zymogen. Measurements of the effect of urea on optical rotation, viscosity, and biological activity of chymotrypsinogen and δ-chymotrypsin are also reported in this communication. These include the effects of time and urea concentration on the measured parameters. On the basis of the data presented, in conjunction with other information on the chemical and physical characteristics of chymotrypsinogen and δ-chymotrypsin, tentative conclusions have been drawn concerning the structural changes involved in activation and denaturation, respectively.

The Activation of Zymogens

Publisher Summary The chapter presents the activation of the zymogens, trypsinogen, chymotrypsinogen, procarboxypeptidase, and pepsinogen. These have been selected because they are among the best characterized representatives of the zymogens as a group. The process of activation of these zymogens has certain common features. Thus the transformations are catalyzed by proteolytic enzymes, which operate with a high degree of specificity and selectivity, affecting the hydrolysis of a limited number of peptide bonds in the zymogen molecule. Since all activation processes considered in this chapter are mediated by proteolytic enzymes, the hydrolytic cleavage of one or more peptide bonds in the zymogen molecule is an essential feature of these reactions. The methods of activation of trypsinogen include activation by Mold Kinase , Enterokinase, and the Autocatalytic activation of trypsinogen. Chymotrypsinogen can be activated by trypsin, and also by the proteinase from Bacillus subtilis (B. subtilis) . The tryptic activation is of greater importance as it is the physiological mechanism, and most of the experimental work has been carried out with this system.

[48] Invertebrate proteases

Publisher Summary This chapter presents a procedure for preparation of crayfish trypsin, hornet chymotrypsin, low-molecular-weight protease (LMWP), and crayfish carboxypeptidase. Crayfish trypsin —together with the two other principal digestive proteases, LMWP and crayfish carboxypeptidase—is synthesized in the hepatopancreas of decapode crustacea and secreted into the cardia, a stomach-like organ from which the enzyme is isolated. Large-scale purification of crayfish trypsin is based on the anion-exchange chromatography and gel filtration. hornet chymotrypsin purification procedure include: extraction by centrifugation; anion-exchange chromatography on diethylaminoethylsephadex A-50. The final purification of fraction VCP II requires gel filtration on Sephadex G-50. LMWP occurs in the cardia extract of crayfish in a concentration of about 1 mg/ml. The digestive juice can be collected from living crayfish by the procedure same as for crayfish trypsin. The purification of crayfish carboxypeptidase is greatly facilitated by use of affinity chromatography on a carboxypeptidase inhibitor from potatoes covalently attached to Sepharose-4B.

Structure, specificity and localization of the serine proteases of connective tissue

It is the purpose of this review to describe several well characterized proteases present in tissues and cells which appear to be related to the pancreatic serine proteases. Much of the early work concerning the nature of tissue proteases was done during the late 19th century by German physiologists who were studying tissue autolysis [ 11. Hedin and Rowland [2] examined homogenates of various mammalian organs for proteolytic activity and observed that, with the exception of muscle, autolysis was greatest at acid pH. The term ‘cathepsin’ was introduced by Willsttitter and Bamann [3] in 1929 to describe proteolytic activity of tissues in the weakly acid pH range. In recent years, however, this term has sometimes been applied to include tissue proteases in general, such as cathepsin G which has an optimum activity at pH 8 and cathepsin E which is most active at pH 2.5. The rapidly growing literature dealing with tissue and cellular proteases has been recently reviewed by Barrett [4]. respiratory and gastrointestinal diseases [4]. Malignant tissues also show significant changes in proteolytic activity compared to normal, particularly that due to collagenases [ 111. As important as abnormal proteolytic activity in tissues may be in disease states, intraand extracellular proteases are necessary for maintaining normal tissue homeostasis. The steady-state concentration of proteins in cells and tissues is controlled by the rates of their synthesis and degradation [ 12,131 and although lysosomal proteases play a major role in intracellular protein degradation [ 141, there is considerable evidence that other proteases are of equal importance [15].