Albert S. Mildvan

ZINC in DNA polymerases

Summary Homogeneous DNA polymerases from E. coli and sea urchins contain zinc in a proportion of approximately 2 and 4 gm atoms Zn/mole enzyme respectively. Specific inhibition of the enzyme by ortho -phenanthroline and lack of inhibition by meta -phenanthroline suggest that the bound zinc plays a functional role in the interaction of DNA with the polymerases.

Mechanisms of signaling and related enzymes

Most enzymes involved in cell signaling, such as protein kinases, protein phosphatases, GTPases, and nucleotide cyclases catalyze nucleophilic substitutions at phosphorus. When possible, the mechanisms of such enzymes are most clearly described quantitatively in terms of how associative or dissociative they are. The mechanisms of cell signaling enzymes range from ≤8% associative (cAMP‐dependent protein kinase) to ≈50% associative (G protein Giα1). Their catalytic powers range from 105.7 (p21ras) to 1011.7 (λ Ser‐Thr protein phosphatase), usually comparable in magnitude with those of nonsignaling enzymes of the same mechanistic class. Exceptions are G proteins, which are 103‐ to 105‐fold poorer catalysts than F1 and myosin ATPases. The lower catalytic powers of G proteins may be ascribed to the absence of general base catalysis, and additionally in the case of p21ras, to the absence of a catalytic Arg residue, which interacts with the transition state. From kinetic studies of mutant and metal ion substituted enzymes, the catalytic powers of cell signaling and related enzymes can be rationalized quantitatively by factors contributed by metal ion catalysis (≥105), general acid catalysis (≈103±1), general base catalysis (≈103±1), and transition‐state stabilization by cationic and hydrogen bond donating residues (≈103±1). Proteins 29:401–416, 1997.

Quantitative interpretations of double mutations of enzymes

The quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on which kinetic or thermodynamic parameter of an enzyme is measured, the same two mutations can interact differently in the double mutant. Additive effects of two mutations on an equilibrium constant, such as the dissociation constant of the enzyme-substrate complex (KS), occur when noninteracting residues which facilitate the same step (substrate binding) are mutated. Partially additive effects result from the cooperative interaction with the substrate of the two residues mutated, and synergistic effects result from the anticooperative interaction with the substrate of the two residues mutated. An alternative explanation for synergy is extensive unfolding of the enzyme. Antagonistic effects on an equilibrium constant such as KS result from opposing structural effects of the two mutations on substrate binding. No additional effect of the second mutation in the double mutant represents a limiting case of either partial additivity or antagonism [corrected]. The interactions of the effects of two mutations on a rate constant such as kcat have the same explanations as those given above for equilibrium constants since the binding of a rate-limiting transition state is occurring. However, due to kinetic complexity, the following exceptions and additions exist. Additive effects of two mutations on kcat occur when noninteracting residues which facilitate the same step are mutated, provided this step is rate limiting. If the affected step is not rate limiting then synergistic effects of the two mutations are observed as each mutation causes the step to become progressively more rate limiting. Additive effects on kcat also occur when the two mutations affect consecutive steps, provided one of them is rate limiting. Partially additive effects on kcat also occur when noninteracting residues facilitating consecutive, non-rate-limiting steps are mutated. These concepts, when applied to published data on double mutants of delta 5-3-ketosteroid isomerase, staphylococcal nuclease, tyrosyl-tRNA synthetase, glutathione reductase, and subtilisin, provide deeper insights into the independent, cooperative, anticooperative, or antagonistic interactions of amino acid residues in the binding of substrates, activators, and inhibitors and in promoting catalysis.

[15] Nuclear relaxation measurements of the geometry of enzyme-bound substrates and analogs

Publisher Summary This chapter focuses on the nuclear relaxation measurements of the geometry of enzyme-bound substrates and analogs. Knowledge of the conformations and arrangement of substrates bound at the active sites of enzymes can yield valuable clues to the mechanism of enzyme action. Measurement of nuclear magnetic relaxation rates in the presence of paramagnetic probes, a nuclear magnetic resonance (NMR) method that determines distances from the individual atoms of a molecule in a solution to a nearby paramagnetic reference point, has emerged in the past decade as a useful approach to the study of the conformation and arrangement of enzyme-bound substrates in a solution. Inactive substrate analogs rather than true substrates are often used by NMR spectroscopists and crystallographers to determine distances and conformations on enzymes because of their desirable spectral properties and because they are not altered by the enzyme reaction. Hence, although competitive analogs are useful, there is no assurance that they provide information directly relevant to the conformation of the bound active substrate.

Mechanistic studies of cAMP-dependent protein kinase action.

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzymes catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

High‐Precision Measurement of Hydrogen Bond Lengths in Proteins by Nuclear Magnetic Resonance Methods

We have compared hydrogen bond lengths on enzymes derived with high precision (≤ ±0.05 Å) from both the proton chemical shifts (δ) and the fractionation factors (ϕ) of the proton involved with those obtained from protein X‐ray crystallography. Hydrogen bond distances derived from proton chemical shifts were obtained from a correlation of 59 O—H····O hydrogen bond lengths, measured by small molecule high‐resolution X‐ray crystallography, with chemical shifts determined by solid‐state nuclear magnetic resonance (NMR) in the same crystals (McDermott A, Ridenour CF, Encyclopedia of NMR, Sussex, U.K.: Wiley, 1996:3820–3825). Hydrogen bond distances were independently obtained from fractionation factors that yield distances between the two proton wells in quartic double minimum potential functions (Kreevoy MM, Liang TM, J Am Chem Soc, 1980;102:3315–3322). The high‐precision hydrogen bond distances derived from their corresponding NMR‐measured proton chemical shifts and fractionation factors agree well with each other and with those reported in protein X‐ray structures within the larger errors (±0.2–0.8 Å) in distances obtained by protein X‐ray crystallography. The increased precision in measurements of hydrogen bond lengths by NMR has provided insight into the contributions of short, strong hydrogen bonds to catalysis for several enzymatic reactions. Proteins 1999;35:275–282.

[29] Nuclear relaxation measurements of water protons and other ligands

Publisher Summary This chapter discusses the way nuclear relaxation rates are measured and only those aspects of the theory that elucidate the methods and their applications. The measurement of the relaxation rates of magnetic nuclei is a specialized branch of nuclear magnetic resonance spectroscopy, which, especially when carried out with paramagnetic probes, can provide thermodynamic, structural, and kinetic information on enzyme complexes. Specifically, the stoichiometry and dissociation constants of binary and ternary complexes of enzymes with paramagnetic ions, paramagnetic substrate analogs, and diamagnetic substrates may be measured. Coordination schemes and interatomic distances between enzyme-bound paramagnetic metal ions or substrate analogs and the substrate molecules have been determined. The exchange rates of substrates into paramagnetic and diamagnetic environments on enzymes have been estimated. By analogy, when a population of magnetic nuclei is placed in a magnetic field, the magnetic vectors experience a torque and precess about the direction of the field. Energy may be applied to this system to align the magnetic vectors of the nuclei to precess in phase with each other.

Inhibition of Reverse Transcription In Vivo by Elevated Manganese Ion Concentration

Mutations in PMR1, a yeast gene encoding a calcium/manganese exporter, dramatically decrease Ty1 retrotransposition. Ty1 cDNA is reduced in pmr1 mutant cells, despite normal levels of Ty1 RNA and proteins. The transposition defect results from Mn(2+) accumulation that inhibits reverse transcription. Cytoplasmic accumulation of Mn(2+) in pmr1 cells may directly affect reverse transcriptase (RT) activity. Trace amounts of Mn(2+) potently inhibit Ty1 RT and HIV-1 RT in vitro when the preferred cation, Mg(2+), is present. Both Mn(2+) and Mg(2+) alone activate Ty1 RT cooperatively with Hill coefficients of 2, providing kinetic evidence for a dual divalent cation requirement at the RT active site. We propose that occupancy of the B site is the major determinant of catalytic activity and that Mn(2+) at this site greatly reduces catalytic activity.

Escherichia coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT family of proteins. Cloning, purification, and characterization of the enzyme.

The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the β-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg2+, a Km of 0.8 mM and a kcat of 5.2 s−1 at 37°C for dATP. dAMP is a weak competitive inhibitor with a Ki of approximately 4 mM, while PPi is a much stronger inhibitor with an apparent Ki of approximately 20 μM. The enzyme contains the highly conserved signature sequence GXVEX2ETX6REVXEEX2I designating the MutT family of proteins. However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT− mutator phenotype, and thus must serve a different biological role in the cell.

Short, strong hydrogen bonds on enzymes: NMR and mechanistic studies

The lengths of short, strong hydrogen bonds (SSHBs) on enzymes have been determined with high precision (^0.05 A ˚ ) from the chemical shifts (d ), and independently from the D/H fractionation factors (f ) of the highly deshielded protons involved. These H-bond lengths agree well with each other and with those found by protein X-ray crystallography, within the larger errors of the latter method (^ 0.2 to ^ 0.8 A ˚ ) (Proteins 35 (1999) 275). A model dihydroxynaphthalene compound shows a SSHB of 2:54 ^ 0:04 Abased on d ¼ 17:7 ppm and f ¼ 0:56 ^ 0:04; in agreement with the high resolution X-ray distance of 2:55 ^ 0:06 A ˚ . On ketosteroid isomerase, a SSHB is found ð2:50 ^ 0:02 AÞ; based on d ¼ 18:2 ppm and f ¼ 0:34; from Tyr- 14 to the 3-O 2 of estradiol, an analog of the enolate intermediate. Its strength is , 7 kcal/mol. On triosephosphate isomerase, SSHBs are found from Glu-165 to the 1-NOH of phosphoglycolohydroxamic acid (PGH), an analog of the enolic intermediate ð2:55 ^ 0:05 AÞ; and from His-95 to the enolic-O 2 of PGH ð2:62 ^ 0:02 AÞ: In the methylglyoxal synthase - PGH complex, a SSHB ð2:51 ^ 0:02 AÞ forms between Asp-71 and the NOH of PGH with a strength of