Barry S. Cooperman

Evolutionary conservation of the active site of soluble inorganic pyrophosphatase

Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism. PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions. Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (Onu) directly attacks PPi with a pKa vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

Dynamics of translation by single ribosomes through mRNA secondary structures

During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

Role of hybrid tRNA-binding states in ribosomal translocation

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steady-state kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (ktrans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases ktrans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3′‐ends of the tRNAs at the peptidyl‐transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl‐transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Protein estimation by the product of integrated peak area and flow rate.

A convenient method for protein estimation is described, making use of uv detectors and peak integrators that are standard equipment on modern high-performance liquid chromatographs to determine the product of integrated peak area and flow rate of eluting protein at 214 nm (AF214). We demonstrate that AF214 is proportional to the amount of eluted protein and describe two approaches for calibrating the integrator, by quantitative amino acid analysis and by determining the elution yield of a known amount of applied protein, allowing direct estimation of protein from AF214. Both approaches yield similar results. The basis for the method is that, for virtually all proteins, absorbance at 214 nm is dominated by the summed contributions from the peptide groups. More accurate estimates can be made when the amino acid composition of the eluting protein is known, since this permits a correction to be made for contributions of amino acid side chains to absorbance at 214 nm. Comparison of AF214 estimates for proteins from the small (30 S) subunit of the Escherichia coli ribosome with those obtained by Bradford analysis shows the latter to give somewhat higher values.

Evolutionary aspects of inorganic pyrophosphatase

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.

Cytoplasmic Inorganic Pyrophosphatase

Pyrophosphate (PPi) is the smallest member of the polyphosphate family and is formed by two phosphate (Pi) residues linked by a phosphoanhydride bond. A specific enzyme hydrolyzing PPi to Pi was discovered in animal tissues in 1928 (Kay) and later in a great many other organisms and cell types, in virtually all in which it has been sought. Its initial name was “pyrophosphatase”, later elongated with a questionable “inorganic”.

Design of optimized high-performance liquid chromatographic gradients for the separation of either small or large molecules : I. Minimizing errors in computer simulations

Abstract Computer simulations can be used to develop high-performance liquid chromatographic gradient elution methods. However, the usefulness of this approach depends on the accuracy of the resulting predictions. Possible sources of error in computer simulation for the prediction of separation based on gradient elution have been investigated. This has in turn led to recommendations for minimizing such errors. With suitable precautions it appears possible to make adequately reliable predictions of separation by gradient elution. Several examples with protein mixtures as samples are reported.