Biochemistry - Chemical Reactions in the Living Cell, Volume 1 - D. Metzler 1980

The molecules we are made of
How we study molecular structure
Hydrolysis

Almost all Biopolymers are inherently unstable and break down into monomer units in the Presence of Water (undergo hydrolysis). Hydrolysis is catalyzed by protons, hydroxyl ions, and Enzymes. The mechanisms of hydrolysis are discussed in Chapter 7. Hydrolysis can be complete or partial, non-specific, or, conversely, targeted at specific bonds within the polymer molecule.

Complete Protein Hydrolysis is typically carried out by heating at approximately 110° in an N₂ atmosphere in the presence of 6 N HCl for 12–96 hours. Some Amino Acids, particularly Tryptophan, are degraded under these conditions. Essentially, a method for achieving ideal complete hydrolysis does not exist¹. Complete protein hydrolysis can also be accomplished using basic catalysts, but this is accompanied by significant Amino Acid Racemization.

Nucleic Acids are likewise hydrolyzed using strong acids. For instance, heating in 12 N perchloric acid at 100 °C for 1 hour is sufficient to cleave the molecule down to its constituent bases. In DNA, N-glycosidic bonds are more labile than in RNA, and purine bonds are more labile than pyrimidine bonds. This characteristic allows for a very useful Procedure: if DNA is left overnight in the cold at pH 2, complete depurination takes place. The resulting polymer is known as apurinic acid (Table 2-11).

Alkaline hydrolysis of RNA yields a mixture of 2'- and 3'-NUCLEOTIDES. The Mechanism of this process involves the participation of free 2'-OH groups of ribose and The formation of cyclic 2',3'-phosphates, closely mirroring the MECHANISM OF ACTION of pancreatic Ribonuclease (Chapter 7, Section D.2). Because deoxyribose lacks a free 2'-OH group, DNA remains unaffected in an alkaline environment.

Enzymatic Methods of hydrolysis are particularly valuable due to the high Specificity they often exhibit. Trypsin, an endopeptidase, rapidly cleaves peptide bonds only when the carbonyl group of the susceptible amide bond belongs to one of the basic amino acids—Lysine or Arginine. Thus, trypsin converts a protein into a relatively small number of tryptic Peptides that can be separated and characterized. Trypsin hydrolyzes only denatured Proteins, and prior Cleavage of disulfide bridges is required to achieve optimal results.

¹ It has recently been reported that superior results are obtained by complete hydrolysis using 4 N methanesulfonic acid in the presence of 3-(2-aminoethyl)indole [131a].

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Table 2-11 Some Hydrolytic Cleavage Reactions of Oligonucleotides³

A. Cleavage at points a is carried out by:

1. Endonucleases (throughout the length of the molecule)

Pancreatic deoxyribonuclease I

2. Exonucleases (from the 3'-end only)

Non-specific snake venom diesterase, attacks both DNA and RNA. The reaction requires the mandatory participation of a free 3'-OH group

B. Cleavage at points b is carried out by:

1. Random cleavage along the entire molecule by endonucleases and alkalis (non-Enzymatic hydrolysis)

Pancreatic ribonuclease cleaves the chain exclusively to the right of pyrimidine-containing nucleotides

Ribonuclease T1 from Aspergillus oryzae cleaves the chain to the right of guanine-containing residues (3'-guanylate)

Ribonuclease T2 from Aspergillus oryzae cleaves the chain to the right of adenine-containing residues (3'-adenylate)

Pancreatic deoxyribonuclease (DNase) II Micrococcal DNase

2. Exonucleases (from the 5'-end only)

Spleen phosphodiesterase from bovine spleen hydrolyzes both polyribo- and polydeoxyribonucleotides

^a The table includes reactions affecting both RNA and DNA

⁶ The circled P symbol here denotes the —PO^-₂— group.

Other Enzymes, such as Chymotrypsin and Pepsin (Chapter 7, Section G.2), are less selective, yet they can still be utilized to cleave the peptide chain into fragments for subsequent structural determination. To establish the complete Amino Acid Sequence of a protein, one must identify "overlapping" fragments containing sequences that bridge the ends of two different tryptic fragments. In this manner, the peptides can be "lined up" in the exact order they occur in the native protein.

Let us now consider the peptide mapping method. Its First stage involves the Cleavage of Disulfide Bonds; the protein is then denatured and hydrolyzed using enzymes such as trypsin or pepsin. This yields a set of peptides whose size and Amino Acid Composition are characteristic of each individual protein. The peptide mixture is applied to a sheet of chromatographic paper and subjected to Chromatography in one dimension and Electrophoresis in the other. The peptides are localized as discrete spots, forming a characteristic pattern (a "fingerprint"). The peptide mapping method is particularly useful for detecting minor differences in Cell/13.html">Protein Structure, such as variations between genetic Variants of the same protein (Fig. 4-20).

Certain enzymes catalyze the sequential removal of amino acids from one or the other end of a peptide chain. Carboxypeptidases cleave amino acids from the carboxyl terminus, whereas aminopeptidases attack the opposite end. Using chromatographic techniques, one can determine which Amino acids have been released by these enzymes at given time intervals, thereby gaining insight into The amino acid sequence at the ends of the chain.

Complete enzymatic Hydrolysis of Proteins is carried out using a mixture of enzymes composed of proteases synthesized by Fungi (pronases). However, the enzymes in this mixture also digest one another, so that enzymatic hydrolysis is generally unsuitable for the quantitative breakdown of a protein into its constituent amino acids. One novel approach relies on The Use of hydrolytic enzymes immobilized on agarose gel columns. The protein to be hydrolyzed is passed through the gel, after which the constituent amino acids accumulate at the bottom of the Column [132].

Exonucleases cleave nucleotides from the ends of polynucleotide chains, whereas endonucleases introduce breaks within the chain. Some of these enzymes hydrolyze only single-stranded molecules, whereas others act on double-stranded ones. Certain Nucleases cleave both DNA strands, whereas others "nick" the molecule by introducing a break into only a single strand. The specificity of several nucleases is summarized in Table 2-11. Partial enzymatic hydrolysis of RNA cleaves the molecule into short nucleotide sequences, enabling the Determination of the complete RNA sequence. (The first RNA whose sequence was established was Alanine tRNA. Cleavage was performed using pancreatic ribonuclease and ribonuclease T1 [133].) Two-dimensional Polyacrylamide gel electrophoresis has proven to be a very useful method for obtaining nucleotide maps [134].

Appendix 2-B

Isotopes in Biochemical Research

Both stable^a and radioactive ^b-g isotopes are widely used in chemical and biological research. The Introduction of isotopic labels revolutionized The Study of METABOLISM. In one of the earliest biological experiments utilizing the stable isotope ¹⁵N (detected by mass spectrometry), Schoenheimer and co-workers in 1937 discovered an unexpectedly high Rate of protein turnover in living Tissues (Chapter 14, Section B). Using ¹⁴CO₂, Calvin et al. first traced the path of carbon during Photosynthesis (Appendix 11-A). Similarly, the use of ³²P and ³⁵S isotopes enabled the study of phosphorus and sulfur metabolism; tritium (³H) has found widespread application for labeling diverse Organic compounds, such as thymine. The use of radioactive isotopes forms The basis of sensitive Analytical Methods, including radioimmunoassay for trace amounts of Hormones (Appendix 16-A). Through Applications such as radioautography, isotopes facilitate numerous analytical investigations (see accompanying photograph) and constitute the basis of a valuable end-group determination method that reveals nucleotide sequences (Fig. 2-37).

Differences in isotopic mass, particularly when moving from ¹H to ²H and subsequently to ³H, often strongly affect reaction rates; studies of kinetic isotope effects based on this phenomenon have provided a deeper understanding of the mechanisms of many enzymatic reactions and all the details of their stereochemistry. A striking example is the synthesis and subsequent utilization of chiral acetate (Chapter 7, Section K.2.g). For these investigations, J. W. Cornforth was awarded the Nobel Prize in 1975^e. The specific properties of isotopes form the basis of NMR spectroscopy (Section I).

Some of the isotopes widely used in biochemistry are listed in the table above. For radioactive isotopes, half-lives are given along with the type of emitted particles and their energy. The rays emitted, for example, during the decay of ¹²⁵I and ¹³¹I possess high penetrating power; their intensity, like that of the strong ß-radiation of the ³²P isotope, is very easy to measure. Another isotope, ³H (tritium), is much more difficult to detect^f, but the weak, short-range ß-particle it emits makes tritium uniquely suited for microautoradiography. Knowing the half-life of a given isotope (Equation 6-4) allows one to determine the amount required to produce a specific decay rate; this is of great practical importance because it enables one to find the number of disintegrations per minute necessary to count pulses with a sufficiently low statistical error. Radioactivity corresponding to 3.7∙10¹⁰ disintegrations per second (the radioactivity of 1 g of pure radium, 0.3 mg of the ³H isotope, or 0.22 g of the ¹⁴C isotope) is defined as one curie (Ci). One millicurie (mCi) equals 2.22∙10⁹ dpm (disintegrations per minute)^-1. A labeled compound typically contains a relatively small amount of the radioactive isotope compared to the stable isotope of the same element. Radioactive preparations are characterized by their total radioactivity, expressed in millicuries or microcuries, and their specific activity, expressed in mCi∙mmol^-1. For example, if a compound contains the ³H isotope at a single specific position and has a specific activity of 50 mCi∙mmol^-1, then 0.17% of that position is occupied by ³H.

Radioautogram illustrating the Separation of Escherichia coli proteins incorporating ¹⁴C-labeled amino acids^d. A 25 µL sample (180,000 cpm) containing ~10 µg of protein was applied to a polyacrylamide gel column (2.5 × 130 mm) and subjected to isoelectric focusing (Section Z.1.e). As a result, the proteins were separated according to their isoelectric points. The gel was then removed from the column and placed onto the edge of a polyacrylamide gel slab. Subsequent perpendicular electrophoresis in a sodium dodecyl sulfate solution resulted in the separation of the protein molecules by size. Over 1,000 spots can be distinguished on the radioautogram, obtained by exposing photographic film to the gel slab for 875 hours.

^a Matwiyoff N. A., Ott D. G., Science, 181, 1125–1132 (1973).

^b Wang C. H., Willis D. L., Loveland W. D., Radiotracer Methodology in the Biological, Environmental and Physical Sciences, Prentice-Hall, Englewood Cliffs, New Jersey, 1975.

^c Wang Y. Ed., Handbook of Radioactive Nuclides, The Chemical Rubber Co., Cleveland, Ohio, 1969.

^d Thornburn C. C., Isotopes and Radiation in Biology, Butterworth, London, 1972.

^e O’Farrell P. H., JBC, 250, 4007–4021 (1975).

^f Cornforth J. W., Science, 193, 121–125 (1976).

^g Bransome E. D., Jr. ed., Liquid Scintillation Counting, Grune and Stratton, New York, 1970.

Methods for determining DNA nucleotide sequences were developed more slowly, yet they are now quite well established [134a]. Cleavage of the DNA molecule near a site with a specific sequence (usually palindromic, Section G.11) is accomplished using specific Restriction Endonucleases (Chapter 15, Section E.1). The resulting fragments are then subjected to depurination or treated with exonucleases [135]. Excellent results are obtained by Cellulose acetate electrophoresis followed by a specialized thin-layer "homochromatography"¹ on DEAE-cellulose [136]. If the oligonucleotides are radiolabeled at one end, the sequence can be determined by partial hydrolysis with an exonuclease (see following paragraphs) followed by electrophoresis and homochromatography (Fig. 2-37). During homochromatography, the shortest oligonucleotide migrates the farthest; each successive spot located below corresponds to a compound containing one more nucleotide than the previous one. From the relative shift of one spot with respect to another during electrophoresis, the approximate nucleotide sequence can be reconstructed [137, 137a]. Thus, in Fig. 2-37, the topmost spot corresponds to an oligonucleotide of unknown composition, and the nucleotide immediately below it contains one additional thymine residue, as clearly indicated by its higher electrophoretic mobility at pH 3.5. The addition of guanine increases electrophoretic mobility to a lesser extent, whereas the addition of adenine has almost no effect on it. Conversely, the presence of an extra cytosine decreases electrophoretic mobility. The sequence can be read directly from the nucleotide map: (5')-TTATTAGCCAGAAGT-(3'). These data were confirmed by further studies involving purine removal and nearest-neighbor analysis. Another recently developed method for rapid DNA Sequencing is described in [137b].

There is a distinct group of specific enzymes that cleave Polysaccharides. Typically, these enzymes are specific for a particular sugar incorporated into the chain via a specific type of glycosidic bond. Examples of this type include starch-hydrolyzing enzymes. Salivary and pancreatic α-amylases cleave starch molecules randomly, whereas plant ß-amylases sequentially release maltose from the ends of unbranched chains (Chapter 7, Section B.6).

¹ The separation of labeled oligonucleotides is carried out in a solvent containing a random mixture of unlabeled nucleotides (RNA hydrolysis products). During plate development, the unlabeled nucleotides occupy the positions of the test labeled nucleotides and migrate along the plate distances roughly corresponding to their sizes.

Complex Lipids are degraded by lipases. Pancreatic lipase removes the 1- and 3-acyl groups of triglycerides to yield 2-monoglycerides. Phospholipase A, present in various tissues and Bacteria as well as in snake venom, selectively cleaves the acyl group at either the 1- or 2-position from Phospholipids. Phosphodiesterases, known as phospholipases C and D, respectively, are present in bacterial and plant tissues; they cleave the chain on opposite sides of the phosphodiester bond, as shown below.

FIG. 2-37. Two-dimensional nucleotide map obtained by partial hydrolysis of a ³²P-oligonucleotide cleaved from a circular DNA copy synthesized on globin mRNA template (Chapter 15, Section J-4). The oligonucleotide was treated with snake venom exonuclease (phosphodiesterase, Table 2-11), which removes nucleotides sequentially from the 3'-end. The topmost spot corresponds to a nucleotide of unknown composition; each subsequent spot located below contains one additional nucleotide unit. Based on the relative mobility of the intermediate products (see text), the sequence was deduced: (5')-TTATTAGCCAGAAGT-(3') [137].

Mild hydrolysis in the presence of basic catalysts is employed for the Selective Cleavage of carboxylate esters (at the positions indicated in the scheme by A₁ and A₂). The resulting phosphodiesters, amides (Sphingolipids), and esters can be separated and identified.