Annual Review of Biochemistry - Volume 66, 1997

A classic approach in biology, both organismal and cellular, is to compare morphologies in order to glean structural and functional commonalities. The comparative approach has also proven valuable on a molecular level. For example, phylogenetic comparisons of RNA sequences have led to determination of conserved secondary and even tertiary structures, and comparisons of protein structures have led to classifications of families of protein folds. Here we take this approach in a mechanistic direction, comparing protein and RNA enzymes.

The aim of comparing RNA and protein enzymes is to learn about fundamental physical and chemical principles of biological catalysis. The more recently discovered RNA enzymes, or ribozymes, provide a distinct perspective on long-standing questions of biological catalysis. The differences described in this review have taught us about the aspects of RNA and proteins that are distinct, whereas the common features have helped us to understand the aspects that are fundamental to biological catalysis. This has allowed the framework that was put forth by Jencks for protein catalysts over 20 years ago (1) to be extended to RNA enzymes, generalized, and strengthened.

Replication protein A [RPA; also known as replication factor A (RFA) and human single-stranded DNA-binding protein] is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic DNA metabolism, including DNA replication, DNA repair, and recombination. RPA homologues have been identified in all eukaryotic organisms examined and are all abundant heterotrimeric proteins composed of subunits of approximately 70, 30, and 14 kDa. Members of this family bind nonspecifically to single-stranded DNA and interact with and/or modify the activities of multiple proteins. In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage). Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell. RPA may also have a role in modulating gene expression.

Bacterial cell division occurs through the formation of an FtsZ ring (Z ring) at the site of division. The ring is composed of the tubulin-like FtsZ protein that has GTPase activity and the ability to polymerize in vitro. The Z ring is thought to function in vivo as a cytoskeletal element that is analogous to the contractile ring in many eukaryotic cells. Evidence suggests that the Z ring is utilized by all prokaryotic organisms for division and may also be used by some eukaryotic organelles. This review summarizes our present knowledge about the formation, function, and evolution of the Z ring in prokaryotic cell division.

Ternary complexes of DNA-dependent RNA polymerase with its DNA template and nascent transcript are central intermediates in transcription. In recent years, several unusual biochemical reactions have been discovered that affect the progression of RNA polymerase in ternary complexes through various transcription units. These reactions can be signaled intrinsically, by nucleic acid sequences and the RNA polymerase, or extrinsically, by protein or other regulatory factors. These factors can affect any of these processes, including promoter proximal and promoter distal pausing in both prokaryotes and eukaryotes, and therefore play a central role in regulation of gene expression. In eukaryotic systems, at least two of these factors appear to be related to cellular transformation and human cancers. New models for the structure of ternary complexes, and for the mechanism by which they move along DNA, provide plausible explanations for novel biochemical reactions that have been observed. These models predict that RNA polymerase moves along DNA without the constant possibility of dissociation and consequent termination. A further prediction of these models is that the polymerase can move in a discontinuous or inchworm-like manner. Many direct predictions of these models have been confirmed. However, one feature of RNA chain elongation not predicted by the model is that the DNA sequence can determine whether the enzyme moves discontinuously or monotonically. In at least two cases, the encounter between the RNA polymerase and a DNA block to elongation appears to specifically induce a discontinuous mode of synthesis. These findings provide important new insights into the RNA chain elongation process and offer the prospect of understanding many significant biological regulatory systems at the molecular level.

The 3′-ends of both prokaryotic and eukaryotic mRNA are polyadenylated, but the poly(A) tracts of prokaryotic mRNA are generally shorter, ranging from 15 to 60 adenylate residues and associated with only 2–60% of the molecules of a given mRNA species. The sites of polyadenylation of bacterial mRNA are diverse and include the 3′-ends of primary transcripts, the sites of endonucleolytic processing in the 3′ untranslated and intercistronic regions, and sites within the coding regions of mRNA degradation products. The diversity of polyadenylation sites suggests that mRNA polyadenylation in prokaryotes is a relatively indiscriminate process that can occur at all mRNA's 3′-ends and does not require specific consensus sequences as in eukaryotes. Two poly(A) polymerases have been identified in Escherichia coli. They are encoded by unlinked genes, neither of which is essential for growth, suggesting significant functional overlap. Polyadenylation promotes the degradation of a regulatory RNA that inhibits the replication of bacterial plasmids and may play a similar role in the degradation of mRNA. However, under certain conditions, poly(A) tracts may lead to mRNA stabilization. Their ability to bind S1 ribosomal protein suggests that poly(A) tracts may also play a role in mRNA translation.

Phospholipids play multiple roles in cells by establishing the permeability barrier for cells and cell organelles, by providing the matrix for the assembly and function of a wide variety of catalytic processes, by acting as donors in the synthesis of macromolecules, and by actively influencing the functional properties of membrane-associated processes. The function, at the molecular level, of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in specific cellular processes is reviewed, with a focus on the results of combined molecular genetic and biochemical studies in Escherichia coli. These results are compared with primarily biochemical data supporting similar functions for these phospholipids in eukaryotic organisms. The wide range of processes in which specific involvement of phospholipids has been documented explains the need for diversity in phospholipid structure and why there are so many membrane lipids.

Molybdenum-containing enzymes catalyze basic metabolic reactions in the nitrogen, sulfur, and carbon cycles. With the exception of the nitrogenase cofactor, molybdenum is incorporated into proteins as the molybdenum cofactor that contains a mononuclear molybdenum atom coordinated to the sulfur atoms of a pterin derivative named molybdopterin. Certain microorganisms can also utilize tungsten in a similar fashion. Molybdenum-cofactor–containing enzymes catalyze the transfer of an oxygen atom, ultimately derived from or incorporated into water, to or from a substrate in a two-electron redox reaction. On the basis of sequence alignments and spectroscopic properties, four families of molybdenum-cofactor–containing enzymes have been identified. The available crystallographic structures for members of these families are discussed within the framework of the active site structure and catalytic mechanisms of molybdenum-cofactor–containing enzymes. Although the function of the molybdopterin ligand has not yet been conclusively established, interactions of this ligand with the coordinated metal are sensitive to the oxidation state, indicating that the molybdopterin may be directly involved in the enzymatic mechanism.

Two X-ray structures of cobalamin (B12) bound to proteins have now been determined. These structures reveal that the B12 cofactor undergoes a major conformational change on binding to the apoenzymes of methionine synthase and methylmalonyl–coenzyme A mutase: The dimethylbenzimidazole ligand to the cobalt is displaced by a histidine residue from the protein. Two methyltransferases from archaebacteria that catalyze methylation of mercaptoethanesulfonate (coenzyme M) during methanogenesis have also been shown to contain histidine-ligated cobamides. In corrinoid iron-sulfur methyltransferases from acetogenic and methanogenic organisms, benzimidazole is dissociated from cobalt, but without replacement by histidine. Thus, dimethylbenzimidazole displacement appears to be an emerging theme in cobamide-containing methyltransferases. In methionine synthase, the best studied of the methyltransferases, the histidine ligand appears to be required for competent methyl transfer between methyl- tetrahydrofolate and homocysteine but dissociates for reductive reactivation of the inactive oxidized enzyme. Replacement of dimethylbenzimidazole by histidine may allow switching between the catalytic and activation cycles.

The best-characterized B12-dependent mutases that catalyze carbon skeleton rearrangement, for which methylmalonyl–coenzyme A mutase is the prototype, also bind cobalamin cofactors with histidine as the cobalt ligand, although other cobalamin-dependent mutases do not appear to utilize histidine ligation. It is intriguing to find that mutases, which catalyze homolytic rather than heterolytic cleavage of the carbon-cobalt bond, can use this structural motif. In methylmalonylCoA mutase a significant feature, which may be important in facilitating homolytic cleavage, is the long cobalt-nitrogen bond linking histidine to the cofactor. The intermediate radical species generated in catalysis are sequestered in the relatively hydrophilic core of an α/β barrel domain of the mutase.

Modification of Ser and Thr residues by attachment of O-linked N-acetylglucosamine [Ser(Thr)-O-GlcNAcylation] to eukaryotic nuclear and cytosolic proteins is as dynamic and possibly as abundant as Ser(Thr) phosphorylation. Known O-GlcNAcylated proteins include cytoskeletal proteins and their regulatory proteins; viral proteins; nuclear-pore, heat-shock, tumor-suppressor, and nuclear-oncogene proteins; RNA polymerase II catalytic subunit; and a multitude of transcription factors. Although functionally diverse, all of these proteins are also phosphoproteins. Most O-GlcNAcylated proteins form highly regulated multimeric associations that are dependent upon their posttranslational modifications. Evidence is mounting that O-GlcNAcylation is an important regulatory modification that may have a reciprocal relationship with O-phosphorylation and may modulate many biological processes in eukaryotes.

d-amino acids have been detected in a variety of peptides synthesized by animal cells. These include opiate and antimicrobial peptides from amphibian skin, neuropeptides from snail ganglia, a hormone from crustaceans, and a constituent of a spider venom. cDNA cloning has shown that at those positions where a d-amino acid is found in the end-product, a normal codon for the corresponding l-amino acid is present. This implies that the d-residues are formed from l-amino acids by a posttranslational reaction. A prototype enzyme catalyzing such a reaction has recently been isolated from the venom of the funnel web spider.

The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virus type-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for origin-specific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5′-3′ DNA helicase and primase activities, and an origin-binding protein with 3′-5′ DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase α-primase, DNA ligase I, and topoisomerase II, are probably also required.

Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.

Ordered protein aggregation in the brain is a hallmark of Alzheimer's disease and scrapie. The disease-specific amyloid fibrils comprise primarily a single protein, amyloid β, in Alzheimer's disease, and the prion protein in scrapie. These proteins can be induced to form aggregates in vitro that are indistinguishable from brain-derived fibrils. Consequently, much effort has been invested in the development of in vitro model systems to study the details of the aggregation processes and the effects of endogenous molecules that have been implicated in disease. Selected studies of this type are reviewed herein. A simple mechanistic model has emerged for both processes that involves a nucleation-dependent polymerization. This mechanism dictates that aggregation is dependent on protein concentration and time. Furthermore, amyloid formation can be seeded by a preformed fibril. The physiological consequences of this mechanism are discussed.

The discovery that mutations in mitochondrial DNA (mtDNA) can be pathogenic in humans has increased interest in understanding mtDNA maintenance. The functional state of mtDNA requires a great number of factors for gene expression, DNA replication, and DNA repair. These processes are ultimately controlled by the cell nucleus, because the requisite proteins are all encoded by nuclear genes and imported into the mitochondrion. DNA replication and transcription are linked in vertebrate mitochondria because RNA transcripts initiated at the light-strand promoter are the primers for mtDNA replication at the heavy-strand origin. Study of this transcription-primed DNA replication mechanism has led to isolation of key factors involved in mtDNA replication and transcription and to elucidation of unique nucleic acid structures formed at this origin. Because features of a transcription-primed mechanism appear to be conserved in vertebrates, a general model for initiation of vertebrate heavy-strand DNA synthesis is proposed. In many organisms, mtDNA maintenance requires not only faithful mtDNA replication, but also mtDNA repair and recombination. The extent to which these latter two processes are involved in mtDNA maintenance in vertebrates is also appraised.

Transposable elements are discrete mobile DNA segments that can insert into nonhomologous target sites. Diverse patterns of target site selectivity are observed: Some elements display considerable target site selectivity and others display little obvious selectivity, although none appears to be truly “random.” A variety of mechanisms for target site selection are used: Some elements use direct interactions between the recombinase and target DNA whereas other elements depend upon interactions with accessory proteins that communicate both with the target DNA and the recombinase. The study of target site selectivity is useful in probing recombination mechanisms, in studying genome structure and function, and also in providing tools for genome manipulation.

This review focuses on two phospholipase activities involved in eukaryotic signal transduction. The action of the phosphatidylinositol-specific phospholipase C enzymes produces two well-characterized second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This discussion emphasizes recent advances in elucidation of the mechanisms of regulation and catalysis of the various isoforms of these enzymes. These are especially related to structural information now available for a phospholipase C δ isozyme.

Phospholipase D hydrolyzes phospholipids to produce phosphatidic acid and the respective head group. A perspective of selected past studies is related to emerging molecular characterization of purified and cloned phospholipases D. Evidence for various stimulatory agents (two small G protein families, protein kinase C, aand phosphoinositides) suggests complex regulatory mechanisms, and some studies suggest a role for this enzyme activity in intracellular membrane traffic.

Clathrin-coated vesicles were the first discovered and remain the most extensively characterized transport vesicles. They mediate endocytosis of transmembrane receptors and transport of newly synthesized lysosomal hydrolases from the trans-Golgi network to the lysosome. Cell-free assays for coat assembly, membrane binding, and coated vesicle budding have provided detailed functional and structural information about how the major coat constituents, clathrin and the adaptor protein complexes, interact with each other, with membranes, and with the sorting signals found on cargo molecules. Coat constituents not only serve to shape the budding vesicle, but also play a direct role in the packaging of cargo, suggesting that protein sorting and vesicle budding are functionally integrated. The functional interplay between the coated vesicle machinery and its cargo could ensure sorting fidelity and packaging efficiency and might enable modulation of vesicular trafficking in response to demand.

The last stage of protein folding, the “endgame,” involves the ordering of amino acid side-chains into a well defined and closely packed configuration. We review a number of topics related to this process. We first describe how the observed packing in protein crystal structures is measured. Such measurements show that the protein interior is packed exceptionally tightly, more so than the protein surface or surrounding solvent and even more efficiently than crystals of simple organic molecules. In vitro protein folding experiments also show that the protein is close-packed in solution and that the tight packing and intercalation of side-chains is a final and essential step in the folding pathway. These experimental observations, in turn, suggest that a folded protein structure can be described as a kind of three-dimensional jigsaw puzzle and that predicting side-chain packing is possible in the sense of solving this puzzle. The major difficulty that must be overcome in predicting side-chain packing is a combinatorial “explosion” in the number of possible configurations. There has been much recent progress towards overcoming this problem, and we survey a variety of the approaches. These approaches differ principally in whether they use ab initio (physical) or more knowledge-based methods, how they divide up and search conformational space, and how they evaluate candidate configurations (using scoring functions). The accuracy of side-chain prediction depends crucially on the (assumed) positioning of the main-chain. Methods for predicting main-chain conformation are, in a sense, not as developed as that for side-chains. We conclude by surveying these methods. As with side-chain prediction, there are a great variety of approaches, which differ in how they divide up and search space and in how they score candidate conformations.

Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is a key enzyme in the synthesis of glucose in the liver and kidney and of glyceride-glycerol in white adipose tissue and the small intestine. The gene for the cytosolic form of PEPCK (PEPCK-C) is acutely regulated by a variety of dietary and hormonal signals, which result in alteration of synthesis of the enzyme. Major factors that increase PEPCK-C gene expression include cyclic AMP, glucocorticoids, and thyroid hormone, whereas insulin inhibits this process. PEPCK-C is absent in fetal liver but appears at birth, concomitant with the capacity for gluconeogenesis. Regulatory elements that control transcription of the PEPCK-C gene in liver, kidney, and adipose tissue have been delineated, and many of the transcription factors that bind to these elements have been identified. Transgenic mice have been especially useful in elucidating the physiological roles of specific sequence elements in the PEPCK-C gene promoter and in demonstrating the key role played at these sites by the isoforms of CAAT/enhancer binding protein in patterning of PEPCK-C gene expression during the perinatal period. The PEPCK-C gene provides a model for the metabolic control of gene transcription.

Due to its presumed role in regulating cellular cholesterol homeostasis, and in various pathophysiological conditions, acyl-coenzyme A:cholesterol acyltransferase (ACAT) has attracted much attention. Cloning the ACAT gene provides the necessary tool to advance molecular studies of this enzyme. The topics reviewed in this chapter include the pathophysiological roles of ACAT, the biochemistry and molecular biology of the ACAT protein and the ACAT gene, and the mode of regulation by sterol or nonsterol agents in mammalian cells. In addition, we present a working model linking the presumed allosteric property of ACAT with cholesterol trafficking into and out of the endoplasmic reticulum.