STRUCTURE AND FUNCTION OF ENZYMES

GEORGE D. MARKHAM, Ph.D., Senior Member; Adjunct Professor of Biophysics and Member of the Graduate Group in Biophysics, University of Pennsylvania

The objectives of this research are to elucidate the structural basis for the enzymatic catalysis of chemical reactions and to utilize this information in the design of novel specific inhibitors. Our focus is on the mechanisms of enzymes that participate in nucleotide biosynthesis, an essential process in all organisms. The catalytic roles of metal ions in such catalytic processes are of particular interest. We are exploring enzyme structure and function by kinetic and spectroscopic techniques, as well as by molecular biological methods. Computational molecular modeling is being used to study the influence of environment on reaction energetics and molecular structure.

S-adenosylmethionine (AdoMet) synthetase catalyzes the only biosynthesis of the major in vivo methylating agent, AdoMet. AdoMet is involved in numerous metabolic functions including regulation of gene expression and the biosynthesis of a multitude of essential metabolites. AdoMet synthesis occurs from ATP and methionine as shown below:

Elucidation of the conformations of enzyme-bound substrates, products and inhibitors is important to understanding the catalytic mechanism of the enzyme and the design of new inhibitors. In order to obtain structural data for enzyme-bound substrates and product, we have used the NMR technique of two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzymebound AdoMet and 5'-adenylylimidodiphosphate (AMPPNP) (1). AMPPNP, an analog of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a non-hydrolyzable NH-link between the b- and g-phosphates. On the other hand, it is a substrate for AdoMet formation during which tripolyphosphate (PPPi) is initially produced (2). AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C_3'-exo and C_4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site using atomic coordinates from the crystal structure of the enzyme·ADP ·P_i (Figure 1). The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and show how the positive charge formed on the sulfur during AdoMet synthesis is stabilized by interaction with a negatively charged aspartate residue.

ROLES OF RESIDUES INVOLVED IN CATALYSIS BY SADENOSYLMETHIONINE SYNTHETASE. TAYLOR

Crystallographic studies of E. coli AdoMet synthetase have shown that the active site contains 4 aspartate residues; each of two aspartates (residues 16 and 271) provide to the active site the sole protein ligand to one of the Mg²⁺ ions, while complementary NMR studies have implicated the other two aspartates (118 and 238) as interacting with methionine. Since the proposed roles of these residues are electrostatic, each of the four aspartate residues has been changed to an uncharged asparagine residue, and the resultant purified enzymes have been extensively characterized. The mutants structurally resemble the wild type enzyme as indicated by circular dichroism spectra and tetrameric structure. However, all are dramatically impaired in their ability to catalyze AdoMet synthesis; they show kcat reductions of 103- to 104-fold, whereas the ATP and methionine Km values change by less than 5fold. In the partial reaction of PPPi hydrolysis, mutants of the Mg²⁺ binding residues have >103-fold reduced catalytic efficiency (kcat/Km), whereas the D118N and D238N mutants are impaired less than 10-fold. Addition of AdoMet improves the catalytic efficiency of the D16N and D271N mutants >100 fold, similar to the improvement found with the wild type enzyme. In contrast, AdoMet reduces the catalytic efficiency of the D118N and D238N mutant enzyme (with methionine in their binding sites) showing that the mutants cannot undergo the conformational change required for activation. None of the mutations have dramatic effects on the concentration dependence for Mg²⁺ activation, which may indicate that the Mg²⁺ ions are more extensively ligated to the polyphosphate chain than to the enzyme.

Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the K⁺-dependent reaction,

     IMP + NAD + H₂O -> XMP + NADH + H⁺,

which is the rate-limiting step in guanine nucleotide biosynthesis. This enzyme has long been a target for drug design. The catalytic mechanism of the human type II IMPDH isozyme has been studied by measurement of the pH dependencies of the normal reaction, its inactivation by the affinity label 6chloropurineribotide (6-Cl-PRT), and the hydrolysis of 2-chloro-IMP, which yields XMP and Cl^- in the absence of NAD (3). The pH dependence of the IMPDH reaction shows the involvement of both acidic and basic groups in catalysis. None of the kinetic pK values correspond to ionizations of the free substrates and, thus, reflect ionization of the enzyme or enzyme-substrate complexes. The rate of inactivation by 6-Cl-PRT, which modifies the sulfhydryl group of the active site residue cysteine331, increases with pH; the pK of 7.5 reflects the ionization of the sulfhydryl in the E·6ClPRT complex. The pKs of the acids observed in the IMPDH reaction likely also reflect ionization of the cysteine-331 sulfhydryl group, which adds to the purine ring of IMP prior to hydride transfer to NAD. The pH dependence of the rate of hydrolysis of 2-Cl-IMP shows a pK value for a basic group, similar to that seen in the overall reaction, but does not exhibit the ionization of an acidic group. This suggests that the active site cysteine is not involved in this reaction. Surprisingly, the rate of 2-Cl-IMP hydrolysis and the rate of inactivation by 6-Cl-PRT are not stimulated by K⁺, in contrast to the effect of this cation in the IMPDH reaction. The molecular interpretation of the results of our kinetic studies will be greatly facilitated by the newly determined crystal structure of the enzyme (4).

Sulfur compounds such as thiols (RSH, where R is an alkyl group), sulfides (RS-R) and sulfonium ions (R3S⁺) play many important roles in biological systems as metabolic intermediates and as constituents of proteins. In protein structures, thiol and sulfide species act as stabilizing agents by participation in disulfide and hydrogen bonds, and as nucleophiles in enzymatic reactions. Sulfonium ions act as alkyl group donors in a variety of biosynthetic reactions. To elucidate the influence of the surrounding environment on the conformations and reactivity of these species, we have conducted extensive high level theoretical studies of representative small molecules, both alone and in aqueous complexes (5, 6). These studies reveal that interactions of the sulfur with surrounding electron rich atoms, such as oxygen, or electron-accepting hydrogen-bond donors, are profoundly influenced by the electronegativity of the R groups attached to the sulfur. An extreme case is provided by the positively charged sulfur of sulfonium ions such as that of AdoMet. The relationships discovered between the geometries of the complexes formed and their energies aid in understanding the environments of sulfur atoms in proteins and in developing force fields for the improved modeling of macromolecular species.

A common role of metal ions in biological systems is to bind a water molecule and activate it for participation in a chemical reaction. The structures and energies of metal-water complexes are intertwined in complex fashions that are not readily understood from experimental measurements. We have used theoretical methods to evaluate the properties of water complexed with a variety of metal ions (5). The influence of other ligand types on the acidity of coordinated water bound to the particularly important Mg²⁺ ion has also been evaluated (6). These type of studies are only now becoming possible due to the explosive increases in computer power and the development of new quantum chemistry methodologies.

1.   SCHALK-HIHI, C., MARKHAM, G.D. The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase. Biochemistry 38:2542-2550, 1999.

2.   RECZKOWSKI, R.S., TAYLOR, J.C., MARKHAM, G.D. The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate. Biochemistry 37:13499-13506, 1998.

3.   MARKHAM, G.D., BOCK, C.L., SCHALK-HIHI, C. Acid-basic catalysis in the chemical mechanism of inosine monophosphate dehydrogenase. Biochemistry 38:4433-4440, 1999.

4.   COLBY, T.D., VANDERVEEN, K., STRICKLER, M.D., MARKHAM, G.D., GOLDSTEIN, B.M. Crystal structure of human type II inosine monophosphate dehydrogenase: Implications for ligand binding and drug design. Proc. Natl. Acad. Sci. U.S.A. 96:3531-3536, 1999.

5.   KATZ, A.K., GLUSKER, J.P., MARKHAM, G.D., BOCK, C.W. Deprotonation of water in the presence of carboxylate and magnesium ions. J. Phys. Chem. B 102:6342-6350, 1998.

6.   TRACHTMAN, M., MARKHAM, G.D., GLUSKER, J.P., GEORGE, P., BOCK, C.W. Interactions of metal ions with water: Ab initio molecular orbital studies of structure, bonding enthalpies, vibrational frequencies and charge distributions. 1. Monohydrates. Inorganic Chem. 37:4421-4431, 1998.

MARKHAM, G.D., BOCK, C.L., TRACHTMAN, M., BOCK, C.W. Intramolecular non-bonded interactions between oxygen and group VIA elements. An ab initio molecular orbital and density functional theory investigation of the structures of HXCH₂CHO (X = S, Se and Te). J. Mol. Struct. (Theochem.) 459:187-199, 1999.

MARKHAM, G.D., BOCK, C.L., TRACHTMAN, M., BOCK, C.W. Intramolecular non-bonded interactions between oxygen and group VIA elements: An ab initio molecular orbital and density functional theory investigation of the structures of HXCH₂COOH (X = S, Se and Te). Struc. Chem. (in press).

Paper in press at time of previous report:

MARKHAM, G.D., TRACHTMAN, M., BOCK, C.L., BOCK, C.W. The binding of water to the carboxylate group in RCO^-₂ (R=H, CH₃, NH₂, OH, and F): an ab initio molecular orbital study. J. Mol. Struct. 455:239-259, 1998.

^§   Fox Chase researcher

^a   C. Schalk-Hihi: Present address-3-D Pharmaceuticals, Exton, PA 19341

^b   C.W. Bock: The Philadelphia College of Textiles and Science, Chemistry Department, Philadelphia, PA 19144

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998