(Professor J.P.G. Malthouse)

Research Group Photographs
 

Inhibition of Proteolytic enzymes (Professor J.P.G. Malthouse)
Humans have a range of proteases which are important for a range of processes including digestion of food, blood clotting, control of blood pressure, etc.  However, there are specific proteases, which can be targeted to treat various diseases, e.g. the AIDS virus needs a protease to multiply, cancers and parasites use proteases to move through tissues, proteases are used to produce the amyloid plaque protein which causes Alzheimer's disease.  Therefore to treat such conditions we need to inactivate the proteases causing the disease.  This inactivation can be achieved using protease inhibitors.   There are four main types of proteases the thiol proteases: the serine proteases, the metalloproteases and the aspartyl proteases.  We usually wish to target just the one protease causing the disease and not inhibit other proteases which are essential for health. We are synthesising protease inhibitors and using NMR to determine how they interact with specific proteases.  We hope that these studies will help us  optimise their ability to inhibit the specific proteases involved in a range of diseases.

Serine Proteases
It is thought that the serine proteases get much of their catalytic efficiency from stabilising the tetrahedral intermediates formed in catalysis.  But because tetrahedral intermediates do not accumulate during catalysis they cannot be directly studied.  One solution to this problem is to study transition state analogues which mimic the tetrahedral intermediates formed during catalysis.  Specific ¹³C-enrichment of such inhibitors allows us to use ¹³C-NMR to identify and characterise a single carbon atom within an enzyme-inhibitor adduct.  In this project we intend to use this approach to study the stabilisation of tetrahedral adducts by the serine proteases by using NMR to determine the pKa values of active site groups. 

A new lysine derived glyoxal inhibitor of trypsin, its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin. Cleary, J.A. & Malthouse, J.P.G. Biochemistry and Biophysics Reports, 2016, 5, 272-284

Hemiacetal stabilisation in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin. Jennifer A. Cleary, William Doherty, Paul Evans, J.Paul G. Malthouse. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 2014, 1844, 1119-1127.

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C-NMR spectra of glyoxal inhibitors. Spink, E., Hewage, C.M. and Malthouse, J.P.G. Biochemistry, 2007, 46, 12868-12874.

13C-and 1H- NMR  studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimising inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide derived chloromethylketone and glyoxal inhibitors. Malthouse, J.P.G.Biochem. Soc. Trans (2007) 35, 566-570

Djurdjevic-Pahl, A., Hewage, C. and Malthouse, J.P.G., Ionisations within a subtilisin–glyoxal inhibitor complex. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics (2005) 1749, 33-41

Djurdjevic-Pahl, A., Hewage, C. and Malthouse, J.P.G A ¹³C-NMR study of the inhibition of d-chymotrypsin by a tripeptide-glyoxal inhibitor
Biochem. J. (2002) 362, 339-347.

MacSweeney, A., Birrane, G., Walsh, M.A., O'Connell, T., Malthouse, J.P.G. and Higgins, T.D.
Crystal structure of d-chymotrypsin bound to a peptidyl chloromethyl ketone inhibitor.
Acta Crystallogr. D Biol Crystallogr (2000) 56, 280-286.

Malthouse, J.P.G. Using Nuclear Magnetic Resonance as a probe of protein structure and function.
National Committee for Biochemistry Award Medal Lecture
Biochem. Soc. Trans. (1999) 27, 701-713.

O' Sullivan, D.B.,  O'Connell, T.P., Mahon, M.M., Koenig A., Milne, J.J., Fitzpatrick, T.P. and Malthouse, J.P.G. A ¹³C-NMR study of how the oxyanion pKa of subtilisin and chymotrypsin tetrahedral adducts are affected by different amino acid residues binding in the enzymes S₁-S₄ subsites. Biochemistry (1999) 38, 6187-6194

O'Connell, T.P. Day, R.M., Torchilia, E.V., Bachovchin, W.W. and Malthouse, J.P.G. (1997) A ¹³C-NMR study of the role of Asn-155 in stabilising the oxyanion of a subtilisin tetrahedral adduct Biochem. J. 326, 861-866.

O'Connell, T.P. and Malthouse, J.P.G. (1996) Determination of the ionisation state of the active site histidine in a subtilisin-chloromethane inhibitor derivative by ¹³C-NMR Biochem. J. (1996) 317, 35-40.

O'Connell, T.P. and Malthouse, J.P.G. (1995) A study of the stabilisation of the oxyanion of tetrahedral adducts by trypsin, chymotrypsin and subtilisin Biochem. J. 307, 353-359

Finucane, M.D. and Malthouse, J.P.G. (1992) A study of the stabilization of tetrahedral adducts by trypsin and chymotrypsin. Biochem. J. 286, 889-900

Malthouse, J.P.G. and Finucane, M.D. (1991) A study of the relaxation  parameters of a ¹³C enriched methylene carbon and  a  ¹³C enriched perdeuteromethylene carbon attached to chymotrypsin. Biochem. J. 280, 649-657.

Finucane, M.D., Hudson, E.A. and Malthouse, J.P.G. (1989) A ¹³C n.m.r. investigation of the ionizations within an a-chymotrypsin inhibitor complex: Evidence that both a-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms. Biochem. J. 258, 853-859.

Mackenzie, N.E., Grant, S.K., Scott, A.I. and Malthouse, J.P.G. (1986) ¹³C NMR study of the stereospecificity of the thiohemiacetals formed on inhibition of papain by specific  enantiomeric aldehydes.Biochemistry  25, 2293-2298.

Malthouse, J.P.G. ¹³C NMR of enzymes. (1986) Progr. Nucl. Magn. Reson. Spectrosc. 18, 1-59.

Malthouse, J.P.G., Primrose, W.U., Mackenzie, N.E. and  Scott, A.I. (1985) A ¹³C NMR study of the ionisations within a trypsin-chloromethylketone inhibitor complex. Biochemistry 24, 3478-3487.

Malthouse, J.P.G., Mackenzie, N.E., Boyd, A.S.F. and Scott, A.I. (1983)  Detection of a tetrahedral adduct in a  trypsin-chloromethyl-ketone specific inhibitor complex by ¹³C NMR. J. Am. Chem. Soc. 105, 1685-1686.

Howe N, Ceruso M, Spink E, Malthouse JPG, pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor. Biochim Biophys Acta. 2011 Oct;1814, 1394-403. 

The thiol proteases  constitute a broad super family of hydrolases that can play an important role in diseases such as bacterial and viral infection, malaria, inflammation, cancer, AIDS, Alzheimer's disease, leukaemias and myocardial infarction . 
Malaria is caused by protozoan parasites of the Plasmodium genus. Plasmodium faciparum is the most virulent and widespread of the malarial parasites. The parasites invade the host red blood cells and use proteases to hydrolyse the host haemoglobin to amino acids which they use as a food source and to maintain osmotic stability. After treatment with cysteine protease inhibitors the parasitic food vacuole becomes swollen with undigested haemoglobin suggesting that cysteine protease inhibitors could be potent antimalarial drugs. The cysteine protease falcipain 3 has been established as a prime targets for the discovery of antimalarial drugs. We have shown that glyoxal inhibitors are extremely potent inhibitors of the cysteine protease papain. Falcipains 3 is a member of the papain family of cysteine proteases and so we want to study how glyoxal inhibitors interact with falcipain 3. It is hoped that these studies will help in the development of drugs to treat conditions such as malaria.

Cleary JA, Doherty W, Evans P, Malthouse JP. (2015) Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteases. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 2015, 1854, 1382-1391.

Dunny E, Doherty W, Evans P, Malthouse JP, Nolan D, Knox AJ. (2013) Vinyl sulfone-based peptidomimetics as anti-trypanosomal agents: design, synthesis, biological and computational evaluation.  J Med Chem. 2013, 56, 6638-50                                             

Lowther, J., Djurdjevic-Pahl, A., Hewage, C. and Malthouse, J.P.G. A ¹³C-NMR study of the inhibition of papain by a dipeptide-glyoxal inhibitor.
Biochem.J. (2002) 366, 983-987.

 

Investigation of the mechanism of inhibition of aspartyl proteases (Professor J.P.G. Malthouse).

The AIDS virus needs a protease to multiply and proteases are used to produce the amyloid plaque protein which causes Alzheimer's disease.  Therefore to treat such conditions we need to inactivate the aspartyl proteases causing the disease.  We usually wish to target just the one protease causing the disease and not inhibit other proteases which are essential for health. In this project we are synthesising new protease inhibitors and trying to optimise their ability to inhibit the aspartyl proteases.  The proteases targeted in treatment of AIDS are aspartyl proteases, and there is currently great interest in developing specific inhibitors of the b- and g-secretases which are aspartyl proteases which are required to produce the amyloid protein which causes Alzheimer's disease.
 
One in 10 people over 65 and about half those over 85 have Alzheimer's disease. These patients live on average for 8 years and it is estimated that they cost American society at least $100 billion per year and result in business losses of 60 billion per year.  Alzheimer's disease must result in similar but proportionally smaller costs to Ireland.  Clearly developing new aspartyl protease inhibitors and optimising their potency and specificity should have great benefits not only for the treatment of Alzheimer's disease but also for the treatment of AIDS, raised blood pressure and many other diseases.

In this study we intend to synthesise a range of inhibitors of the aspartyl proteases with different structures (alcohol, keto, hydrate etc.) at the catalytic site. We will assess the inhibitor potency (Ki values) of the synthesised inhibitors. We will use NMR to determine how  these inhibitors interact with the aspartyl proteases and assess whether there is maximal interaction with inhibitor carbonyl oxygen, the oxyanion or its conjugate acid.  It is hoped that these studies will give us insights into how to develop inhibitors, which have optimal specificity and inhibitor potency for the aspartyl proteases.
 

An NMR study of the inhibition of pepsin by glyoxal inhibitors: Mechanism of tetrahedral intermediate stabilization by the aspartyl proteases. Cosgrove, S. Rogers, L. Hewage, C.M. and Malthouse, J.P.G. Biochemistry, 2007,  46,11205-15.

Studies on the catalytic efficiency and stereospecificity of a-proton exchange reactions catalysed by pyridoxal phosphate dependent enzymes and catalytic antibodies (Professor J.P.G. Malthouse)
The catalysis of the exchange of the a-proton of amino acids is a common mechanistic feature of most pyridoxal phosphate dependent enzymes which determines the enantiomeric purity of the product.  We are using NMR to undertake a systematic and quantitative study of the factors which affect the stereospecificity of these exchange reactions.  We have developed an NMR-kinetic technique (Malthouse et al., 1991) which allows us to quantify how allosteric effectors, cofactors, enzyme subunits, the substrate a-carboxylate group and different substrate R-groups contribute to the catalytic efficiency and stereospecificity of a-proton exchange reactions catalysed by tryptophan synthase (Malthouse et al., 1991; Bailey & Malthouse 1991; Milne & Malthouse 1995; 1996), serine hydroxymethyltransferase (Malthouse et al., 1991; Fitzpatrick & Malthouse 1998) and the catalytic antibody 15A9 (Mahon et al., 1998).

We are currently using this approach to study a range of pyridoxal phosphate dependent catalysts including aspartate aminotransferase (Professor J.P.G. Malthouse and Professor Philip Christen), catalytic antibodies (Professor J.P.G. Malthouse and Professor Philip Christen) and serine hydroxymethyltransferase (Professor J.P.G. Malthouse and Professor V. Schirch)

Toth, K., Amyes, T.L., Richard, J.P., Malthouse, J.P.G. and NiBelliu, M.E (2004) Claisen-Type Addition of Glycine to Pyridoxal in Water. J.Am. Chem. Soc. 126, 10538-10539

NiBelliu, M.E. and Malthouse, J.P.G. (2004) The stereospecificity and catalytic efficiency of the tryptophan synthase catalysed exchange of the a-protons of amino acidsBiochem. J. 381, 847-852.

Malthouse, J.P.G. (2003). Stereospecificity of a-proton exchange reactions catalysed by pyridoxal-5'-phosphate dependent enzymes.
Biochem. Biophys. Acta, 1647, 138-142.

The aspartate aminotransferase-catalysed exchange of the a-protons of aspartate and glutamate: The effects of the R386A and R292V mutations on this exchange reaction.
Mahon, M.M., Graber, R., Christen, P. and Malthouse, J.P.G. Biochem. Biophys. Acta (1999) 1434, 191-201.

Mahon M., Gramatikova S.I., Fitzpatrick T.B., Christen P. and Malthouse J.P.G. (1998) The pyridoxal-5'-phosphate-dependent catalytic antibody 15A9: Its efficiency and stereospecificity in catalysing the exchange of the a-protons of glycine FEBS Lett. 427, 74-78.

Fitzpatrick, T.B. and Malthouse, J.P.G. (1998) A substrate induced change in the stereospecificity of the serine hydroxymethyltransferase catalysed exchange of the a-protons of amino acids. Eur. J. Biochem. 252, 113-117.

Milne, J.J. and Malthouse, J.P.G. (1996) The effect of different amino acid side chains on the stereospecifity and catalytic efficiency of the tryptophan synthase catalysed exchange of the alpha-protons of amino acids Biochem. J. 314, 787-791

Milne, J.J. and Malthouse, J.P.G.(1995) Factors affecting the stereospecifity and catalytic efficiency of the tryptophan synthase catalysed exchange of the pro-2R and pro-2S protons of glycine Biochem. J.  311, 1015-1019

Malthouse, J.P.G., Milne, J.J. and Gariani, L.S. (1991) A comparative study of the kinetics and stereochemistry of the serine hydroxymethyltransferase and tryptophan synthase catalysed exchange of the pro-2R and pro-2S protons of glycine.Biochem. J. 273, 807-812

Bailey, C.J. and Malthouse, J.P.G. (1991) A Proton Magnetic Resonance Study of hydrogen exchange reactions of Yeast tryptophan synthetase. Biochem. J. 273, 605-610

Using ¹³C-NMR to assess the environment of thiocyanate carbons in proteins
Cyanylation of a cysteine residue to produce a b-thiocyanatoalanine residue provides a simple and economical method of introducing a small ¹³C-enriched reporter group into a protein (Malthouse, 1986; Doherty et al., 1992).  We have used this approach to study changes in the environment of thiols when cyanylated apoflavodoxins bind FMN (Doherty et al., 1992;1993) and to determine the provenance of the thiol group of ß-lactoglobulins A and B (Phelan & Malthouse, 1994).  We have also shown that the thiocyanate carbon of native cyanylated ß-lactoglobulin has a chemical shift of 109.7 ppm while that in samples of unfolded ß-lactoglobulin has a chemical shift of 114.4 ppm (Phelan & Malthouse 1994).  Therefore denaturation can  easily be followed by ¹³C-NMR.

ß-lactoglobulins (Professor J.P.G. Malthouse)
These proteins are found in the milk of cows and they bind a range of ligands tightly including Vitamin A.  They may have an important role in transferring Vitamin A from the mother to its offspring.  In this project we intend to use ¹³C-NMR to determine the stability of cyanylated ß-lactoglobulins under a range of conditions both in the presence and absence of ligands.

Malthouse, J.P.G. and Phelan, P. (1995) The effect of magnetic field strength on the linewidth and spin -lattice relaxation time of the thiocyanate carbon of cyanylated ß-lactoglobulin B; optimisation of the experimental parameters for observing thiocyanate carbons in proteins Biochem. J 306, 531-535

Phelan, P. and Malthouse, J.P.G. (1994) ¹³C- n.m.r. of the cyanylated ß-lactoglobulins: evidence that cys-121 provides the thiol group of ß-lactoglobulins A and B. Biochem.J. 302, 511-516

Flavodoxins
(Professor Mayhew and Professor J.P.G. Malthouse)
We have cyanylated both the thiols of flavodoxin from Megasphaera elsdenii and have observed signals at 109 ppm and 115 ppm which changed on removing FMN (Doherty et al., 1992).  We are currently preparing mutants where one or the other cysteine residues are converted into serine residues. This will allow us to assign the thiocyanate signals in the wild type protein.  This small flavodoxin
(Mr 15000) has been well characterised by X-ray crystallography and so it is a good model for evaluating the sensitivity of thiocyanate carbons to their environments.  Therefore it is planned to make mutants with cysteine residues introduced into various locations which have different hydrophobicities and polarities.  These mutants will enable us to determine how accurately the chemical shift of the thiocyanate carbon reflects its protein environment.
 

Yalloway, G.N., Mayhew, S.G., Malthouse, J.P.G., Gallagher, M.E. and Curley, G.P. pH-dependent Spectroscopic Changes Associated with the
Hydroquinone of FMN in Flavodoxins. Biochemistry (1999) 38, 3753-3762

Doherty, G.M., Mayhew, S.G. and Malthouse, J.P.G. (1993) ¹³C-n.m.r. of the cyanylated apoflavodoxin and flavodoxin from Clostridium pasteurianium  Biochem. J. 294, 215-218.

Doherty, G.M., Motherway, R., Mayhew, S.G. and Malthouse, J.P.G. (1992) ¹³C NMR of cyanylated flavodoxin from Megasphaera elsdenii  and of thiocyanate model compounds.Biochemistry 31, 7922-7930

Synthesis and biosynthesis of isotopically enriched compounds and enzyme inhibitors (Professor J.P.G. Malthouse and Dr K.E. O'Connor)
We have and are synthesising a range of ¹³C-enriched protease inhibitors.  These include chloromethane, glyoxal and ketonic inhibitors.  We have also synthesised doubly enriched(¹³C & ¹⁵N) L-serine and L-tryptophan for NMR studies (Malthouse et al., 1997).

Malthouse, J.P.G., Fitzpatrick, T.B., Milne, J.J., Grehn, L. and Ragnarsson, U. (1997) Enzymatic synthesis of isotopically labelled serine and tryptophan for application in peptide synthesis J. Peptide Science 3, 361-366.

Biotransformation of halophenols using crude cell free extracts of Pseudomonas putida F6.Brooks, S. J., Doyle, E.M., Hewage, C., Malthouse, J.P.G., Duetz, W. and O'Connor, K.E. Appl Microbiol Biotechnol  (2004) 64, 486-492.

Determining how carbon dioxide binds to azacryptand scaffolds

Wild AA, Fennell K, Morgan GG, Hewage CM, Malthouse JPG. (2014), A (13)C-NMR study of azacryptand complexes. Dalton Trans. 2014 43:13557-62 

Properties of LuxF from Photobacterium leiognathi

Bergner T, Tabib CR, Winkler A, Stipsits S, Kayer H, Lee J, Malthouse JP, Mayhew S, Müller F, Gruber K, Macheroux P. (2015) Structural and biochemical properties of LuxF from Photobacterium leiognathi. Biochim Biophys Acta. 2015;1854:1466-1475.