Program

Mary C. O'Sullivan and Qibing Zhou 

Department of Chemistry, Indiana State University, Terre Haute, Indiana 47809 

Trypanosoma cruzi is a protozoan parasite causing Chagas disease which currently infects 16-18 million people leading to more than 45,000 deaths each year. The disease occurs predominantly in Central and South America, however about 100,000 people in the USA are also infected, probably due to transfusion of blood products originating from South America. Trypanothione reductase (TR) is an enzyme that plays a crucial role in the antioxidant defenses of trypanosomes. TR is a NADPH-dependent, FAD-containing enzyme which reduces the disulfide group of N',N'-bis(glutathionyl)spermidine (trypanothione). It has been proposed that inhibitors of TR may have potential as antitrypanosomal drugs. 

In order to develop inhibitors of TR that are synthetically readily available, we initially investigated the inhibiting effects of simple trypanothione analogs, specifically N1‚N3-bis- substituted spermidine derivatives. The results we obtained led us to synthesize and investigate the inhibiting effects of N-substituted spermidines and several substituted spermine derivatives. Several of the polyamine derivatives studied were competitive inhibitors of T. cruzi TR. The most effective compounds investigated were N',N'-bis(2-naphthylmethyl)spermidine, N1‚N®-bis(2- naphthylmethyl)spermine and N,N'-bis(3-phenylpropyl)spermine (K; values of 9.5, 5.5 and 3.5  μM, respectively) with potencies of a similar magnitude to the most effective competitive inhibitor described previously (clomipramine, K; value of 6.5 μM). 

C.J. Anderson, J. Freeman, M. Farley, Linda J.H. Lucas, and Theodore S. Widlanski.

Department of Chemistry, Indiana University, Bloomington, IN 47405 

The enzyme-catalylize desulfation of steroids is a transformation that plays an important role in steroid metabolism. Conversion of steroid sulfates to unconjugated steroids provides a source of steroids for processes such as fertilization, steroid transport, and breast cancer. To define steroid sulfatase's ability to distinguish charged species with geometries similar to sulfate esters such as phosphate esters or their derivatives, we synthesized dehydroepiandrosterone phosphate, dehydroepiandrosterone phosphofluoridate, estrone phosphate, and estrone phosphofluoridate and examined the effect of each compound on the inhibition of steroid sulfatase activity as a function of pH. Estrone phosphate and dehydroepiandrosterone phosphate were found to be potent inhibitors of steroid sulfatase activity at pH 6.0 and pH 5.5 with Ki values of 140 nM and 120 nM respectively. The effect of pH on inhibition of steroid sulfatase activity indicates that the enzyme preferentially binds to the monoanion form of these phosphate esters. Both estrone phosphofluoridate and dehydroepiandrosterone phosphofluoridate had approximately the same effect on steroid sulfatase activity at pH 6.0 and 7.5. A binding mode for these inhibitors based on a transition state analogy (or alternately a ground state bisubstrate mimic) is proposed and incorporated into a model for the mechanism of action of this enzyme. Non-steroidal phosphate inhibitors and substrates have been evaluated. These molecules help to delineate structural features important in enzyme:substrate recognition and indicate that substrates and inhibitors need not have the B, C, and D rings of a steroid, providing that they partition well into the membrane. This finding suggests that the enzyme may utilize substrates other than steroid sulfates. (This work was supported in part by NIH grant R01 GM45572-02, by the Department of the Army grant AIBS 1322, a A.P. Sloan Fellowship and a Camille-Dreyfus Teacher-Scholar Award to TSW). 

Catherine Luschinsky Drennan, Sha Huang, Rowena G. Matthews, and Martha L. Ludwig. 

Biophysics Research Division and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109 

Although the x-ray structure of vitamin B12 (cyanocobalamin) was determined in 1956 by Dorothy Hodgkin, Jenny Glusker, and co-workers, no structure of a B12-protein complex has been available until now. The x-ray analysis of a B12-containing fragment of methionine synthase has revealed motifs and interactions responsible for the recognition of the cofactor (Drennan et al., Science 266, 1669-1674, 1994). Upon binding to methionine synthase, methylcobalamin undergoes a dramatic conformational change in which the dimethylbenzimidazole swings away to open the lower face of the corrin to coordination by a histidine residue from the protein. The 'base-off' cobalamin is bound at the interface of a two-domain structure: a helical domain caps the top face of the corrin and protects the methyl ligand, and an a/ẞ domain interacts with the lower face of the corrin and the displaced dimethylbenzimidazole. The unexpected replacement of the dimethylbenzimidazole with a histidine ligand from the protein has profound mechanistic implications. For methionine synthase, we suggest that the histidine759 ligand, along with neighboring residues aspartate757 and serine810, form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group. 

Supported in part by NIH grants GM 16429 and GM 24908. 

Matthew M. Benning1, Frank M. Raushel2, and Hazel M. Holden1 

1Institute for Enzyme Research, Graduate School and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705 

2Department of Chemistry, Texas A&M University, College Station, Texas 77843 

Phosphotriesterase is a organophosphate-degrading enzyme capable of detoxifying both widely used pesticides such as paraoxon and parathion and various nerve agents. The keat values for the best substrates approach 10 s1 at 25 °C. The enzyme requires a binuclear metal center for activity. We have solved the structures of both the apo- and holo- forms of the enzyme to 2.1 and 2.0 Å resolution, respectfully. The overall fold is an a/ẞ-barrel but there are significant differences in the tertiary architecture of the apo- and holoproteins such that their a-carbon positions superimpose with a root-mean-square deviation of 3.4 Å. The binuclear metal center is located at the C-terminus of the b-barrel with the cadmium atoms separated by 3.8 Å. Unexpectedly, there are two bridging ligands to the metal: a water molecule and a carbamylated lysine residue. The more buried cadmium is surrounded by His 55, His 57, Lys 169, Asp 301 and the bridging water in a trigonal bipyramidal arrangement. The second metal is coordinated in a distorted octahedral geometry by His 201, His 230, Lys 169, the bridging water molecule, and two additional solvents. Comparisons of the apo- and holo- forms as well as inhibitor studies will be discussed. 

Vinod Subramaniam, Nils Bergenhem, Ari Gafni and Duncan Steel 

University of Michigan, Institute of Gerontology, 300 North Ingalls Building, Ann Arbor, MI 48109. 

We recently reported(1) that the structural rigidity of the hydrophobic core of Escherichia coli alkaline phosphatase (AP), measured by recovery of tryptophan phosporescence lifetime, returns to its characteristic native-like value over a period of several days following renaturation in vitro of the extensively (GuHCl-) denatured enzyme. In contrast, the enzymatic activity reaches its asymptotic value in one hour at 24°C. Moreover, the global protein lability, measured by the rate of inactivation in 4.5M GuHCl, also increases on a time scale much longer than the recovery of activity. In the context of the rugged energy landscape model of Frauenfelder et al.(2), the slow annealing of the hydrophobic core is consistent with the presence of high energy barriers that separate fully active intermediates along the folding pathway. These results clearly demonstrate that although the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active, conformation, the phosphorescence data indicates otherwise. This leads to the intriguing conclusion that what is commonly defined as the "native" state may only be a fully active intermediate on the pathway to folding to a more rigid conformation of the protein, which may be nearer the global thermodynamic minimum. [Supported by NIA AG09761; ONR N00014-91-J-1938] 

1. V. Subramaniam, N. C. H. Bergenhem, A. Gafni, D. G. Steel, Biochemistry 34, 1133- 36 (1995). 

2. H. Frauenfelder, S. G. Sligar, P. G. Wolynes, Science 254, 1598-1603 (1991). 

James M. Willard and Ralph A. Gardner-Chavis, 

Cleveland State University, Cleveland, OH 44115

Catalysis by metal ions appears the result of low-lying electronic states of metal ions. The spectra of such electronic states are tabulated in the Atomic Energy Level tables. Such spectra form the basis for our quantitative description of the instant of catalysis by matching two distinct sets of numbers: a perturbation fraction (PF) and an optimum fraction (OF). The PF expresses the inductive effect of the metal ion on adsorbates. The OF reflects the extent of perturbation of two reactants necessary for their reaction. The OF results from the simultaneous solution of pairs of equations which describe the vibrational frequencies of the reactants as functions of their number of electrons. When an OF equals a PF, that metal catalyzes the reaction. The decomposition of hydrogen peroxide by catalase may involve one of the following: (1) OOH <-> O+ OH; (2) OOH <-> H+O2 ; (3) HOOH <-> H2O + O or (4) HOOH <-> H2 + O2. Our analysis suggests only reaction (1) occurs with Fe** removing the exo oxygen or Co** removing either the exo or endo oxygen. Distinction between these two mechanisms by other techniques would be most difficult due to rapid equilibrium.

Ruma Banerjee

Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664 

Methylmalonyl-CoA mutase catalyzes the reversible isomerization of methylmalonyl- CoA to succinyl-CoA in a reaction that is dependent on the cofactor, coenzyme B12 (or AdoCbl), for activity. The reaction involves the 1,2 interchange of a carbonyl CoA group and a hydrogen atom. The first, unifying step in all AdoCbl-dependent reactions is believed to be homolysis of the Co-C bond to generate a pair of radicals-one centered on the cobalt and the other on deoxyadenosine. The details of the migration reaction itself are unclear, and a variety of hypotheses have been extended including a carabonium ion-, free radical-, carbanion-, and an organocobalt adduct as the rearranging species. We have examined, by EPR and other spectroscopic methods, the nature of the bound cofactor in both the ground state and in the presence of substrate. Our studies reveal, that the mutase binds its cofactor in a base-off conformation in which the intramolecular base, dimethylbenzimidazole is replaced at the lower axial position by a histidine residue from the protein. This was predicted based on the sequence homology between the mutase and methionine synthase in the region of the coordinating histidine residue. 15N enrichment of the protein alters the superhyperfine structures in the EPR spectrum and together with DEPC inhibition data, indicates the presence of a histidine ligand. CD spectroscopy provides additional evidence for an altered conformation of bound versus free AdoCbl. 

In the presence of substrate, a 2-component EPR spectrum is obtained with apparent g values of 2.11 and 2.14. Hyperfine coupling to the cobalt nucleus is observed, indicating that the cobalt contributes to the signal. In the presence of perdeuterated substrate, the EPR spectrum is altered indicating that a substrate (or product)-derived radical is a component of the observed signal. In addition, the intensity of the 2.14 but not the 2.11 signal is diminished in the presence of deuterated substrate. ESEEM spectroscopy reveals the presence of coupled matrix protons, and other stronger couplings which are assigned to nitrogens of the coordinating histidine. Our data are consistent with the interpretation that the observed EPR signal is due to a biradical intermediate in which one of the radicals is on the cobalt and the other is organic. We have examined the effect of varying magnetic field on the reaction under steady-state conditions. Unlike ethanolamine ammonia lyase, a B12-dependent enzyme which shows a magnetic field dependence, the reaction catalyzed by the mutase is insensitive to magnetic field, at least in the range tested. In addition, despite substantial deuterium isotope effects on the mutase-catalyzed reaction(DV/K = 4.9, DV = 6.5), a magnetic isotope effect is not seen. The significance of these results in the context of a radical pair mechanism will be discussed 

Kafryn W. Lieder, Weiming Wu, Frank J. Ruzicka, George H. Reed, and Perry A. Frey

Department of Biochemistry, Institue for Enzyme Research, University of Wisconsin, 1710 University Avenue, Madison, Wisconsin 53706. 

EPR spectroscopy has been used to examine organic radicals of substrate and substrate analogues that arise in the steady state of the reaction catalysed by lysine-2,3-aminomutase from Clostridium SB4. Previous EPR studies, using lysine and isotopically substituted forms of lysine, demonstrated the presence of a substrate radical and permitted its characterization as a π-radical with the unpaired spin residing mostly in a p-orbital of C2 of B-lysine. EPR spectroscopy has now been used to examine organic radicals produced when the enzyme is incubated with substrate analogues. These analogues have provided the first method for detecting a B-radical. One analogue, 4,5-dehydrolysine, can stabilize a B-radical as an allyl species. Aminoethylcysteine contains a thioether, which can stabilize a B- radical by orbital overlap with the p-orbitals of the sulfur. EPR spectra gathered from [3-2H2]-aminoethylcysteine indicate that the radical orbital is centered on C3. 

Supported by Grant No. DK 28607 from NIDDK, USPHS, Grant No. GM35752 from NIGMS, NIH, and CMB Training Grant No. GM07215 from NIH. 

Paul Carey 

Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935 

The potential of Raman spectroscopy to provide novel insights into the molecular basis of catalysis has been recognised for some time 1, however, this potential is only now being realised due to technical innovations which make the technique more widely applicable We will provide an overview of progess in relating reactivity to precise structural and electron density information on enzyme-substrate complexes. The following areas will be covered: 

• Acyl-serine proteases - very precise information on acyl C=O bond lengths and strength of C-O-to- oxyanion hole hydrogen bonds. 

• Acyl-cysteine proteases - role of electric field, from a-helix dipole, in stabilizing charge build-up in transition state. 

• Crotonase (enoyl-CoA hydratase) - detection of strong localized electron polarization effects. 

• Dehalogenase - massive electron rearrangement in bound product. 

By undertaking physical chemical/organic studies on compounds which model the active site species it is possible to quantitate many of the above effects and achieve precise structure-reactivity profiles. 

1. P.R. Carey and H. Schneider, J. Mol. Biol.102:679-693 (1976). 

2. M. Kim, H. Owen, and P.R. Carey, Appl. Spectrosc. 47:1780-1783 (1993). 

3. P.R. Carey and P.J. Tonge, Acc. Chem. Res. 28:8-13 (1995).