Zymogenicity


This is a revision of a post that I wrote  several years ago.  Since that time I completed a revision of Chemical Reagents for Protein Modification (1) and, more recently an article on thrombin in the interstitial space (2) and a book (3) on proteolytic enzymes in the interstitial space.   While working on the book on proteolysis in the interstitial space, I became impressed by the large number of proteolytic enzymes and the variety of mechanisms for control of  their activity including  activation from zymogens.    While a majority of these proteases are derived from zymogen forms through the action of another protease, there are a number which are derived from precursor or zymogen forms by autoactivation.   I had worked on pepsin a number of years ago (4) and was thus familiar with the process of autoactivation.   In the case of pepsinogen, acidification promotes the expression of the proteolytic activity of pepsinogen (5,6).  . This work benefited by earlier work by Ong and Perlman (7) who showed that pepsinogen underwent a conformational change on acidification prior to peptide bond cleavage.    These observations and many others  support the concept that a proenzyme or zymogen has a low level of activity as shown by the pancreatic zymogens (8)  This  review by the late Hans Neurath is an excellent but seldom-quoted article on limited proteolysis and zymogen activation. 

   
While wandering in the literature searching for a possible method for thrombin formation (prothrombin activation) in the interstitial space, I came across a paper describing a plasma hyaluronan-binding serine protease (PHBSP) where there is autoactivation of the zymogen (12).  I was also introduced to the term, zymogenicity which is the  ratio of the activity of the active enzyme to that of the zymogen or precursor form ( Aenzyme/Azymogen).  This term is not extensively used (I found 13 citations on PubMed and slightly more on Scifinder®) and I was unaware of its use.  A bit of research showed that the term zymogenicity was first used to describe the latent state of chitin synthase in a variety of fungi and yeasts (13-15).  There has been some recent work on modulating the zymogenicity of tPA(16) and factor X (17) where the goal is an improved therapeutic product.   The zymogenicity of native tPA is approximtely 6.7 (16) which can be increased by protein engineering to greater than 100 (16,18).   Assuming that reactivity with DFP is a reasonable assessment of activity, the zymogenicity of trypsin is on the order of 103  while that of chymotrypsin is on the order of 105 (8).   The zymogenicity of the PHBSP (12) is approximately 400.   As a bit of an aside, the term zymogen (from the Greek, zymo meaning ferment, a term used for enzymes at the time, and gen, meaning derived from) was introduced by Heidenhain in 1985 (19).   Considering present use, it is somewhat strange that Heidenhain used the term zymogen to describe a  proenzyme for amylase (ptyalin).   Subsequent early work on the presence of  zymogens included more work on amylase (20) and trypsin (21).   The paper by Latimer and Warren (20) is well worth reading as the last paragraph describes the frustration of investigators when equivocal results are obtained; it is unlikely that such honesty would be expressed by an author  today and less likely to be tolerated by an editor.


References
1.  Lundblad, R.L., Chemical Reagents for Protein Modification, CRC Press/Taylor and Francis, Boca Raton, Florida, 2014.
2.   De Ridder, G., Lundblad, R.L., and Pizzo, S.V., Actions of thrombin in the interstitium, J.Thromb.Haemost., in press, 2016.
3.  Pizzo, S.V., Lundblad,R.L., and Willis, M.E., Proteolysis in the Interstitial Space, CRC Press/Taylor and Francis, Boca Raton, Florida, expected July, 2016.
4.  Lundblad, R.L. and Stein, W.H.,  On the reaction of diazoacetyl compounds with pepsin, J.Biol.Chem. 244, 154-160, 1969.
5.  Bustin, M. and Conway-Jacobs, A, Intramolecular activation of porcine pepsinogen, J.Biol.Chem. 246, 615-620, 1971.
6.  Kageyama, T., Analysis of the activation of pepsinogen in the presence of protein substrates  and estimation of the intrinsic proteolytic activity of pepsinogen, Eur.J.Biochem. 176,. 543-549, 1988.
7.  Ong, E.B. and Perlmann, G.E., The amino-terminal sequence of porcine pepsinogen, J.Biol.Chem. 243, 6104-6109, 1968.
8.  Neurath, H., Limited proteolysis and zymogen activation, in Proteases and Biological Control., ed  E.Reich, D.B. Rifkin, and E. Shaw, pps. 51-64, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1975.
9. Robinson, N.C., Neurath, H., and Walsh, K.A., The relation of the α-amino group of trypsin to enzyme function and zymogen activation, Biochemistry 12, 420-426, 1973.
10.  Pasternak, A., Liu, X., Lin, T.-Y., and Hedstrom, L., Activating a zymogen without proteolytic processing: Mutation of Lys 15 and Asn194 activates trypsinogen, Biochemistry 37, 16201-16210, 1998.
11.  Bode, W., Schwager, P., and Huber, R., The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 Å resolution, J.Mol.Biol. 118, 99-112, 1978.
12.  Etscheid, M., Hunfeld, A., Kὅnig, H., Seitz, R., and Dodt, J., Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular autocatalytic mechanism,  Biol.Chem. 381, 1223-1231, 2000.
13.   Bartnicki-Garcia, S., Bracker, C.E., Reyes, E., and Ruiz-Herrera, J. Isolation of chitosomes from taxomically diverse fungi  and synthesis of chitin microfibrils in vitro, Expt.Mycology  2, 173-192, 1978.
14.  Haenseler, E., Nyhlen, L.E., and Rast, D.M., Isolation and properties of chitin synthetase from Aganicus bisporus mycelium, Expt.Mycology 7, 17-30, 1983.
15.  Gozalbo, D., Dubón, F., and Sentandreu, R., Studies on zymogenicity  and solubilization of chitin synthase from Candida albicans, Microbiol.Lett. 26, 59-63, 1985.
16.  Tachias, K. and Madison, E.L., Converting tissue-type plasminogen activator into a zymogen, J.Biol.Chem. 271, 28749-28752, 1998.
17.  Ivanciu, L. and Camire, R.M., Hemostatic agents of broad applicability produced by selective tuning of factor Xa zymogenicity, Blood 126, 94-102, 2015.
18.  Madison, E., Kobe, A., Gehring, M.J., Sambrook, J.F., and Goldsmith, E.J., Converting tissue plasminogen activator to a zymogen: a regulatory triad of Asp-Ile-Ser, Science 262, 419-421, 1993.
19.  Mendel, B., Professor Ruldolph Heidenhain, Science 6, 645-648, 1897.
20.  Latimer, C.W. and Warren, J.W., On the presence of the amylolytic ferment and its zymogen in the salivary glands, J.Exp.Med. 2, 465-473, 1987.
21.  Bellamy, J.F., On the agents concerned with the production of the trypsin ferment from its zymogen, J.Physiol. 27, 323-331, 1901.
©Roger L. Lundblad, Chapel Hill, North Carolina, January, 2016