T. Ramasarma

The active site of an enzyme protein occupies a relatively small area of its large surface. Many more activities can be accomplished by a single protein provided more active sites can be fitted on its vast unused (?) surface. This makes sense in the best interest of the cellular economy. The concept of multifunctionality of some enzyme proteins is accepted¹; never mind the dogma of their specificity, as the two sites can act independently. It is not necessary that the same molecule of the protein does one job at a time. There are many molecules of the protein in the cell, and portions of these can be assigned for each task in different parts of the cell. The concept of multiple functions of a protein can be expanded to encompass non-catalytic structural roles. In order to popularize the phenomenon, attempts are made to add colloquial adjectives such as moonlighting and promiscuous. I believe that the use of inappropriate descriptions for catching the attention of readers is likely to be counterproductive. A case is made here for caution and restraint.

One protein – many functions

I had earlier made an attempt to put together some information available on proteins having more than one function in an article in Current Science². The introductory paragraph and the two concluding paragraphs therein, which define succinctly this concept, are reproduced below:

 

‘The concept of one enzyme-one activity has influenced biochemistry for over half a century. Over 1000 enzymes are now described. Many of them are highly "specific". Some of them are crystal-lized and their three-dimensional structures determined. They range from 12 to 1000 kDa in molecular weight and possess 124 to several hundreds of amino acids. They occur as single polypeptides or multiple-subunit proteins. The active sites are assembled on these by appropriate tertiary folding of the polypeptide chain, or by interaction of their constituent subunits. The substrate is held by side-chains of a few amino acids at the active site on the surface, occupying a tiny fraction of the total area. What is the bulk of the protein behind the active site doing? Do all proteins have only one function each? Why not a protein have more than one active site on its large surface? Will we discover more than one activity for some proteins? These newer possibilities are emerging and are find-ing experimental support. Some proteins purified to homogeneity using assay methods for different activities are now recognized to have the same molecular weight and a high degree of homology of amino acid sequence. Obviously they are identical. They represent the phenomenon of one protein – many functions.

‘Three distinct ways of using a protein become apparent from the examples available: multiple catalytic sites, binding properties and structural roles. Thus, a protein active as an enzyme can be "recruited" to do other functions. It can bind to its own substrate or other metabolites (e.g. tRNA) and transport them. Complexing with distinct structures in mRNA or DNA, it can play a regulatory role. The regulatory elements and transcription factors may turn out to be a known enzyme protein. It can become a structural element like in lens crystallins or
in the subunit or membrane structures. Indeed, the possibilities are legion.

‘One other perspective is emerging. How valid is the enzyme nomenclature in the light of these developments? In the foregoing examples, it can be seen that a 58 kDa-protein is identified with at least four distinct catalytic activities, and there are at least two distinctive 58 kDa-proteins with other activities. It is

Table 1. One protein – many functions (1p–nf)

Serial No.	Primary identity	Other activities
1	Protein disulfide isomerase (58 kDa)	– Prolyl hydroxylase (a subunit) – Thyroxine-binding protein – Peptide-binding (sequence non-specific) – Oligosaccharyl transferase (a subunit) – Triglyceride transfer protein (a subunit) – Thioredoxin-like activity
2	Pyruvate kinase (58 kDa)	T3-binding protein
3	Lipid-transfer protein (58 kDa)	Sterol carrier protein 2
4	Fermodulin (58 kDa, HMGCoA reductase inhibitor)	Fe-binding protein (low affinity)
5	Peptidyl-prolyl cis-trans isomerase (17 kDa)	– Cyclophilin – Cyclosporin A-binding protein
6	Glyceraldehyde-3-phosphate dehydrogenase (37 kDa) (a house keeping enzyme, occurs in large concentration)	– Acyl phosphatase – Esterase – ADP-ribosylation – Microtubule-binding protein – Protein kinase – Uracil-DNA glycosylase – t-RNA-binding protein(sequence specific) – Amyloid protein (Amy c)-binding protein – Membrane binding protein
7	Lens crystallins
	– alpha	– Chaperone-like activity6
	– delta	– Arginine-succinate lyase
	– epsilon	– Lactate dehydrogenase B
	– eta	– Aldehyde dehydrogenase
	– lambda	– Hydroxyacyl CoA dehydrogenase
	– mu	– Ornithine cyclodeaminase
	– rho	– NADPH-quinone reductase
	– tau	– Enolase
	– sigma	– GSH-S-transferase
8	Aconitase (with Fe-S cluster, mitochondrial, also in cytosol)	Iron-response element (IRE)-binding protein (no Fe cluster) (coexists in cytosol with active aconitase form)
9	Isocitrate dehydrogenase
	– Mitochondrial, NAD-specific	– Mitochondrial mRNA-binding protein
	– Cytosolic, NADP-specific	– NADH-decavanadate reductase
10	Lactoferrin (Fe-binding, 80 kDa)	Ribonuclease
11	a -subunit of F1-ATPase (mitochondrial, 57 kDa)	Heat-shock protein in peroxisomes
12	Peroxidase (Mn-dependent, N. crassa protein)	Heat shock protein
13	Separate non-overlapping active sites
	– Pseudomonas syringe protein	– Oxygenase and dioxygenase
	– Mitochondrial signal peptidase	– Two catalytic subunits of non overlapping specificities
	– Amylase/Trypsin inhibitor	– Independent inhibition of two activities
14	Leukocyte antigen CD 38 (46 kDa)	– NAD-glycohydrolase
		– ADP-ribose cyclase
		– Cyclic ADP-ribose hydrolase
15	Put A proline dehydrogenase(plasma membrane)	DNA-binding transcriptional repressor in cytoplasm
16	Phosphoglucose isomerase (cytosolic)	Neuroleukin, autocrine motility factor, differentiation and maturity mediator (extracellular)
17	Thymidine phosphorylase (cytosolic)	Platelet-derived growth factor (PDGF) of endothelial cells (extracellular)
18	Carbinolamine dehydratase (converts 4a -hydroxy H4- to quinonoid H2-biopterin)	Dimerization cofactor (DCoH) of hepatic nuclear transcription factor (HNF-1a )
19	Thioredoxin of E. coli	T7 -DNA polymerase subunit (heterodimeric)
20	Aspartate receptor of E. coli	Maltose-binding protein receptor (different binding site)
21	PMS2 mismatch repair enzyme	Blood cell protein for hypermutation of antibody variable chain
22	Cystic fibrosis transmembrane conductance channel regulator (CFTR), cAMP-dependent Cl- channel	Epithelial sodium channel regulator protein
23	Multidrug resistance transmembrane transporter (MDR ATPase, p-glycoprotein)	Cell swelling ion channel regulator protein
24	Thrombin (protease in blood clotting cascade)	Ligand for cell surface receptor, PAR-1 (G-protein coupled)
25	Thymidylate synthetase	Binding protein of own mRNA, inhibits translation
26	Biotin-5¢ -adenylate synthetase (E. coli)	Repressor protein of Bio operon
27	LON protease (ATP-dependent) (mitochondrial)	Mitochondrial chaperone
28	Ftsh protein (assists protein transport across membranes in bacteria)	Metalloprotease (ATP-dependent)
29	Afg 31/Rcalp protein (facilitates assembly of F0F1-ATPase)	Protease, degrades improperly folded proteins
30	Band 3 protein (RBC plasma membrane)-anion exchanger	Glycolysis inhibitor protein (acts by N-terminal domain binding to aldolase, glyceraldehyde-3-phosphate dehydrogenase and phosphofructokinase
31	L-Aspariginase	Nitrilase (3-diazo-4-oxo-L-norvaline ®  N2)
32	A-Esterase	Phosphotriesterase
33	Carbonic anhydrase-III	Esterase, phosphotriesterase, phospho monoesterase (P-tyrosine preferred)
34	Chymotrypsin	Phosphotriesterase; acylation of own His57 (from p-nitrobenzene sulfonate)
35	Cytosine methyltransferase	Cytosine deamination
36	Myoglobin	Sulfoxidation of thioanisole
37	Pepsin A	Phenyl sulfite hydrolysis
38	Phytase (an acid phosphatase)	Sulfoxidation (vanadate-dependent peroxidation reaction)
39	Serum albumin (general binding protein)	Esterase (p-nitophenyl acetate substrate)
40	Urease	Phosphoramidate hydrolysis
41	Adenylate kinase	Sulfuryl transfer from ADP-sulfate to acceptor
42	Alkaline phosphatase	Sulfatase; phosphodiesterase (substrates: sulfate and phosphate esters of p-nitrophenol)
43	Arylsulfatase A	Cyclic phosphodiesterase (cAMP hydrolysis)
44	Aspartate aminotransferase	b-Elimination
		– sulfate from L-serine-O-sulfate
		– chloride from b-chloro-L-alanine
		– b-carboxyl group of aspartate
45	Pyruvate oxidase	Acetohydroxy acid synthetase
46	Chloroperoxidase (vanadium-dependent)	Phosphomonoesterase (p-nitrophenyl phosphate hydrolysis)
47	RNA-binding enzymes7
	– Catalase	– own mRNA
	– Dihydrofolate reductase	– own mRNA
	– Glutamate dehydrogenase	– mRNA of cytochrome oxidase
	– Lactate dehydrogenase	– homopolymeric RNA
48	Lectin (V. faba seeds)8	a -Galactosidase
49	Non-histone protein BA9	Glutathione-S-transferase
50	Apoprotein B-100 of LDL10	Phospholipase A2
51	H+-ATPase (reticulocyte endosomes)11	Iron-binding protein
52	G-protein bg-subunits (HEK 293 cells)12	MAP kinase activator
53	DNAase (bovine pancreatic)13	Phosphatase (intrinsic; not phosphodiesterase)
54	Lipoamide dehydrogenase (part of pyruvate dehydrogenase complex-NAD specific)14	NADPH-ubiquinone reductase (Zn-acivated)
55	Cytochrome P450 2C (coronary arteries)15	Endothelium-dependent hyperpolarization factor (EDHF) synthase
56	Hemoglobin (ascaris)16	Nitric oxide activated dioxygenase
Pooled information available on alternative functions of some proteins: 1–14 from ref. 2; 15–30 from ref. 3; 31–45 from ref. 4 (original references are given in these three reviews).

probably time to consider developing a "protein nomenclature" based on domain- level amino acid sequence, structu-ral motifs and their arrangements in polypeptides. More and more proteins
will now be identified with multiple functions’.

The above article appeared to be well received in India by the number of references to the simple, expressive (but not catchy, colloquial) title ‘One protein – many functions’ that I noticed in the meetings and private discussions. A modest number of reprint requests came from several countries, not unusual in these days of easy photocopying. This may reflect non-availability of the journal. The first paragraph reproduced above was available in abstracting services, and was accessed by a scientist in the UK while requesting the reprint. Thus this information is readily available via the Internet to the interested workers and journals. At the end of this article was introduced a cartoon of Ravana with ten faces – a multifaceted personality!

Is a protein having a second function, a moonlighter?

An article entitled ‘Moonlighting proteins’ by Jeffrey³ appeared in TIBS under the section ‘Talking Points’. This article, with no reference to the two previous reviews on the subject^1,2, attracted
my attention as it dealt with multiple functions of proteins. The theme was similar in that a protein can have more than one function as given in their introductory paragraph: ‘cells have had to develop sophisticated mechanisms for switching between the distinct functions of these proteins’. The author clearly implied either/or type of action, made obvious by the example: ‘The aconitase/IRE-BP protein is either an enzyme or an RNA-binding protein, not both simultaneously’. But there are many molecules of this protein, some in mitochondrial matrix acting as aconitase and some in the cytosol acting as IRE-BP. The two forms coexist, but in different parts of the cell and function simultaneously². The cartoon in the TIBS article clearly shows the scientist doing different jobs – inside the laboratory during the day and outside by night. This seemed to have prompted the author to describe them as ‘moonlighting proteins’. I checked the dictionary for what ‘moonlighter’ means. The word was developed originally to describe ‘one of a band of cowardly ruffians in Ireland in 1880 who committed agrarian outrages by night’!

A mild protest letter was sent to the editor, TIBS stating thus: ‘I am perplexed at the title . . . certainly not the way to describe a protein having two or more functions. The cell recruits such proteins for these multiple roles deliberately . . . in the best interest of the economy of the cell’. The editor promptly responded first by e-mail and then by a letter: ‘moonlighting is a colloquial term for holding two jobs – often (but not essentially) one of these at night. The definition you have found in your dictionary has largely passed out of common usage, at least in the UK. We try and check that colloquial words and phrases as understood by our readers worldwide and obviously failed this time’. Thanking him for the prompt response, I reiterated: "the word used is unsuitable to describe the phenomenon. Different molecules of the same polypeptide do the assigned multiple tasks – all the time (not by day or night)’. Interestingly The Times of India (Bangalore, 26 March 1999) published a story with a cartoon on ‘a constable who moonlighted as a robber off duty’. The newspaper cutting was sent to the editor, TIBS indicating that ‘Bangalore is fairly contemporary . . . . Indeed an example is produced for a moonlighter’. The editor, forwarded my letter and the reprint of the Current Science article to the author, and after that I heard nothing. Authors tend to be silent in such situations. Given a break in time, issues are silenced. Silence is golden.

 

Is an enzyme having a second catalytic activity, promiscuous?

Another article by O’Brien and Herschlag⁴ discussed ‘catalytic promiscuity of enzymes’. Again, the dictionary defines the word, promiscuous, as ‘mixed, confused, indiscriminate, not restricted to one’. But promiscuity is usually associated with ‘sexual intercourse . . . without order or distinction’. (I dread the follow-up words/synonyms – whore, pimp, lewd, harlot). What a way to describe a ‘good’ protein serving the cell with multiple chores? It almost implies the second activity (whichever it is) is undesirable. This does not seem to be the intention of these authors, as they described the phenomenon from the limited viewpoint of evolutionary acquisition of an additional activity by a protein ‘for creation of new metabolic pathways from enzymes that were capable of accepting a wide range of related substrates’. They introduced ‘catalytic promiscuity’ to describe ‘enzymes with an ability to catalyse multiple chemical transformations that are normally classified as different types of reactions’. In simple words, one protein – many functions, described earlier². But this article did not benefit from the previous reviews on the subject^1–3, as none of them is found in their reference list. They summarized the primary and the so-called ‘promiscuous’ activities of several proteins in three tables: (1) ‘some active sites can catalyze seeming disparate reactions’; (2) ‘enzymes . . . found to have a low level of an alternative activity . . . of an evolutionarily related enzyme’; (3) ‘examples of increased promiscuous activity from protein engineering’. The theme developed by these authors is that ‘catalytic promiscuity could have aided the evolution of new enzymes via divergent evolution’.

The cell uses a polypeptide in many ways

The three articles^2–4 dealt with the theme that a polypeptide can be used in more than one way in the cell. Trying to draw the attention of readers, the authors^3,4 have used over-reaching personification of an enzyme protein that puts it in a bad perspective. A passing reference to such names may tickle the reader but continuing the appellation is unfair. Some damage is already done. A recent report⁵ called the cell-junction forming protein, pinin, a moonlighting protein (without acknowledging ref. 3) based solely on its dual location in the cell! Restraint is needed by the authors, and caution by the editors.

Information in these reviews on multiple functions of some proteins is pooled and given in Table 1 along with some additions.

One protein – many aliases

Another dimension is added to the problem of naming a protein: one protein-many aliases. Entitled ‘Wanted: A new order in protein nomenclature’, the editorial in Nature (1999, 401, 411) pointed out that ‘A single protein is often studied simultaneously by a number of independent laboratories, each using their own pet name and refusing to acknowledge other names or agreeing to accept a single label’. The protein, Eph B2 receptor, is an example. It has tyrosine kinase activity and is involved in signalling in the brain. It is also known by many aliases – Cek, Nek, Erk, Qek5, Tyro6, Sek3, Hek5 and Drt – according to species and tissues studied, and functions identified by different workers. It is the same protein described from many angles (remember the elephant and the blind men, with apology). The Nature editorial pleaded for stating all other known names of a protein for the first time it is mentioned in the text, and then proceed to describe the function under study. A systemization of the protein nomenclature is indeed wanted. In my article², the question of validity of the enzyme nomenclature was raised and a suggestion for developing a ‘protein nomenclature’ was made. Perhaps this would be realized soon.

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New bugs in science reporting

In the contemporary scene, scientists find it very useful to have articles, such as trends, perspectives, hypotheses, opinions, talking points and minireviews. These make it easy to understand the racing developments in a particular field. These articles require a good deal of reading and thought on the part of the writer. They provide a ready means to understand some emerging phenomena. They often enlighten the readers and enable relating these to their own work. It is understandable such articles greatly influence readers, and earn the journals high impact factors. A departure from simple, sober, prosaic writing in science towards catchy titles, mind-boggling appellations, attention-freezing illustrations and even slogan-worthy dogmas has become trendy. Do we really need to emulate newspaper reporting in science journals? In the wake of their high visibility, there is also a lurking danger that some of them can mislead with diversions into unproductive activities. Watch-out –colloquialism is here.

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T. Ramasarma is in the Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.