Biochemical Genetics, Vol. 15, Nos. 1/2, 1977

Relationship Between Enzyme Heterozygosity and Quaternary Structure Robert D. Ward 1

Received 18 May 1976--Final 30 July 1976

The need for proteins to maintain particular quaternary structures constrains variability in amino acid sequence. Monomeric enzymes are then expected to be more variable than dimeric forms, which in turn are expected to be more variable than tetrameric forms. These predictions are confirmed by analysis of available data on enzyme variation. Theories relating enzyme heterozygosity to metabolic function are discussed in the light o f these findings.

KEY WORDS: enzyme polymorphism; enzyme structure; enzyme function. INTRODUCTION Most natural populations of plant and animal species are genetically variable for an appreciable fraction of their protein loci (reviewed by Lewontin, 1974). Two or more alleles coding for products of differing charge exist at many loci, and this variability may be detected using electrophoretic techniques. Populations that appear to be essentially monomorphic usually either are of small size or have passed through a bottleneck of numbers in the recent past (Avise and Selander, 1972; Bonnell and Selander, 1974). It has become clear that some enzymes are, on average, more variable than others, and it is important to our understanding of the processes of molecular evolution that the reasons for this be explored. Correlations have been sought between enzyme function and heterozygosity (Gillespie and Kojima, 1968; Kojima et al., 1970; Johnson, 1971, 1974; Powell, 1975), but so far the influence of enzyme structure has not been examined. Recent Financial support for part of the work described in this article was derived from NERC Grant GR3/1558 to J. A. Beardmore. 1 Department of Genetics, University College of Swansea, Swansea, United Kingdom. 123 © 1977 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. N o part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.

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124 Table 1. Enzyme Heterozygosity"'b

Enzyme

Structure

ADH ODH XDH TO AO ctGPDH 6PGDFI MDH IDH G6PDH SDH LDH ME G3PDH

Dimer Dimer Dimer Dimer Dimer Dimer Dimer Dimer Dimer Dimer Tetramer Tetramer c Tetramer

AK HK CK PGM GOT

Monomer Monomer Dimer Monomer Dimer

EST LAP PEP ADA AMY ALP ACP

c Monomer --c Monomer Monomer Dimer Dimer

ALD FUM

Tetramer Tetramer

PGI TPI

Dimer Dimer

Group

Invertebrate mean heterozygosity per locus-+ sE

Oxidoreductases NG 0.188 + 0.039 NG 0.118 + 0.028 NG 0.162 + 0.044 NG 0.069 _+0.028 NG 0.250 + 0.053 G 0.028 _ 0.0! 2 G 0.016_+0.007 G 0.063 _+0.017 G 0.127 _+0.026 G 0.179 _+0.045 NG -G -G 0.140 _+0.032 G 0.111 _+0.040 Transferases NG 0.185_+0.039 G 0.081 _+0.019 NG -G 0;247_+0.033 G 0.037 _+0.031 Hydrolases NG 0.241 _+0.021 NG 0.193 _+0.029 NG -NG -NG 0.224 _+0.091 NG 0.109 _+0.025 NG 0.171 _+0.035 Lyases G 0.020 + 0.006 G 0.069 -+0.027 Isomerases G 0.210 + 0.045 G 0.130 + 0.039

Vertebrate mean heterozygosity per locus + sz

0.057_+ 0.025 0.011 _+0.010 0.010 + 0.006 0.038 -+0.014 -0.061 _+0.015 0.081 _+0.019 0.020 _+0.006 0.051 _+0.013 0.022 + 0.020 0.023 + 0.018 0.023 _+0.006 0.085 + 0.029 0.000 + 0.000 0.018+0.013 -0.000 _+0.000 0.119+0.017 0.051 _+0.013 0.141 0.005 0.059 0.281

_+0.016 _+0.005 _+0.020 _+0.084 -0.058 + 0.048 0.055 + 0.032 0.029 -+0.020 0.000-+ 0.000 0.051 _+0.013 --

"Abbreviations: G, glucose-metabolizing enzyme; NG, non-glucose-metabolizing enzyme; ADH, alcohol dehydrogenase; ODH, octanol dehydrogenase; XDH, xanthine dehydrogenase; TO, tetrazolium oxidase (indophenol oxidase) ; AO, aldehyde oxidase; ~xGPDH, c~-glycerophosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; MDH, malate dehydrogenase; IDH, isocitrate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; SDH, sorbitol dehydrogenase; LDH, lactate dehydrogenase; ME, malic enzyme (NADP-malate dehydrogenase); G3PDH, glyceraldebyde-3-phosphate dehydrogenase; AK, adenylate kinase; HK, hexokinase; CK, creatine kinase; PGM, phosphoglucomutase; GOT, glutamate oxalate transaminase (aspartate aminotransferase); EST, esterase; LAP, leucine aminopeptidase; PEP, peptidase (excluding LAP); ADA, adenosine deaminase; AMY, amylase; ALP, alkaline pbosphatase; ACP, acid phosphatase; ALD, aldo-

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studies on rates of protein evolution have led to the proposal that functionally less important molecules or parts of molecules evolve more rapidly than more important ones (Dickerson, 1971; Kimura and Ohta, 1974). It follows that multimeric enzymes are expected to have slower evolutionary rates than monomeric enzymes of similar subunit size, since multimeric proteins will have critical regions required for the maintenance of quaternary structure that will be lacking in monomeric enzymes. Amino acid substitutions in these regions are likely to be selected against (Dickerson, 1971). Studies on protein heterozygosity and rates of protein evolution are but two sides of the same coin, and thus such arguments also predict that monomeric enzymes will be more variable than dimeric enzymes, which in turn will be more variable than tetrameric forms. The present article relates heterozygosity to quaternary structure using available data on enzyme polymorphisms. The findings are discussed with reference to the neutralist/selectionist schools, and with reference to possible relationships between enzyme variation and metabolic function. M A T E R I A L S AND M E T H O D S Data from invertebrate and vertebrate species are considered separately. Parthenogenetically reproducing animals are excluded, as are plant species which are not yet sufficiently well characterized for a test of the hypothesis to be made. Only species for which 15 or more protein loci had been typed in 15 or more individuals were considered in this survey. Enzymes screened in fewer than five invertebrate or vertebrate species were omitted, thus reducing the chances of estimating totally inaccurate mean heterozygosities for some enzymes. The average heterozygosity at one locus in a single population of a species is the observed number of heterozygotes divided by the sample size. These values were calculated, and the mean heterozygosity per species per locus was estimated by averaging over all sampled populations. Averaging these values gave the overall mean heterozygosities per enzyme given in Table I. In no lase; FUM, fumarase; PGI, phosphoglucose isomerase (phosphohexose isomerase) ; TPI, triosephosphate isomerase. b Invertebrate data are taken from 27 Drosophila species and 14 other invertebrate species. Vertebrate data are taken from 13 fish species, 6 amphibian species, 19 lizard species, 5 bird species, and 26 mammal species. Sources of data are given in the references section. ~' c ME, PEP, and invertebrate EST have variable quaternary structure and are not included in any discussion of quaternary structure. The great majority of vertebrate ESTs behave as monomers and are so classified for the purposes of analysis. See text ['or a further discussion of these points.

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instance was weighting for sample size carried out. When only allele frequencies were given, the proportion of heterozygotes was estimated from Hardy-Weinberg expectations. In a few instances, a particular allele appears to give rise either to no product or to an inactive product (e.g., Selander and Yang, 1969). The present article does not distinguish this mode of variation from that detected by alterations of enzyme mobility, and the latter type of variation is itself frequently associated with quantitative changes in activity or in other kinetic aspects (Lewontin, 1974). Since null alleles are rare, they contribute little to the overall heterozygosity estimates. In this survey, catalytically similar enzymes such as the esterases are lumped under a single heading. This is inevitable, since homologies over species are difficult to determine for many of the enzymes coded for by multiple gene loci. A similar approach has been taken by a number of other authors (Johnson, 1974; Powell, 1975), but it does pose certain problems. One, particularly relevant here, is the possibility that in certain instances nonhomologous but catalytically similar enzymes may have differing quaternary structure. For example, although leucine aminopeptidase is invariably a monomer, some of the other peptidases behave as monomers and others as dimers following electrophoresis (Lewis and Harris, 1967). Likewise, some malic enzymes seem to behave as monomers (Hedgecock and Ayala, 1974; Tracey et al., 1975), others as dimers (cited in Manwell and Baker, 1970), and yet others as tetramers (Selander et al., 1969, 1971). Esterases in invertebrates behave sometimes as monomers, sometimes as dimers. These enzymes cannot be considered in the tests of quaternary structure. Vertebrate esterases seem to form a more consistent group than those of invertebrates: those detected through the use of "orthodox" naphthyl substrates and whose gel patterns were described in the articles covered in this survey behave as monomers. On the other hand, it appears that those vertebrate esterases specific for the "unorthodox" umbelliferyl esters behave as dimers (human esterase D, Hopkinson et al., 1973; plaice esterase D, Ward, unpublished) and are thus excluded from the analysis. Human red blood cell acid phosphatase is believed to have a monomeric structure rather than the more usual dimeric structure of acid phosphatase (Hopkinson et al., 1963) and is also excluded. It should be possible to consider supernatant and mitochondrial forms separately, but in fact this cannot be done here since many surveys do not state the intracellular locations of the enzymes studied, and grouping only those forms known to be homologous would entail a great loss of useful information. However, provided that the enzyme groups used in testing the hypothesis have a consistent quaternary structure, and care has been taken to ensure that this is so, the lumping together of nonhomologous but catalytically similar enzymes should not bias the results in any systematic way. The

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q u a t e r n a r y s t r u c t u r e s g i v e n in T a b l e I a g r e e w i t h t h o s e t a b u l a t e d , f o r m a n , b y H o p k i n s o n et al. (1976). A l l s u r v e y s o f w h i c h I a m a w a r e t h a t s u p p l i e d d a t a in a s u i t a b l e f o r m f o r a b s t r a c t i o n w e r e u s e d i n t h e p r e p a r a t i o n o f t h i s article. T h i s i n c l u d e s , f o r e x a m p l e , t h e s t u d y o n t h e e l e p h a n t seal b y B o n n e l l a n d S e l a n d e r (1974). T h i s s p e c i e s w a s f o u n d t o b e t o t a l l y i n v a r i a n t a t all t h e e n z y m e loci a s s a y e d . R e s u l t s like t h e s e a r e e x p e c t e d t o r e d u c e a n y d i f f e r e n c e s in h e t e r o z y g o s i t y a m o n g m o n o m e r i c , d i m e r i c , a n d t e t r a m e r i c e n z y m e classes.

RESULTS Mean heterozygosities per enzyme locus for both invertebrate and vertebrate

Table II. Numbers of Species Investigated Invertebrates

Vertebrates

Enzyme

Loci

Number of species

Genera

Loci

Number of species

Genera

ADH ODH XDH TO AO c~-GPDH 6PGDH MDH IDH G6PDH SDH LDH ME G3PDH AK HK CK PGM GOT EST LAP PEP ADA AMY ALP ACP ALD FUM PGI TPI

27 33 25 41 22 34 16 59 30 18 --35 12 26 47 -34 16 133 52 --6 33 43 22 20 19 20

25 30 23 33 18 34 11 41 28 18 --28 11 12 23 -28 12 39 35 --6 21 24 21 19 17 17

1 4 6 14 2 9 5 14 12 6 --10 8 7 9 -13 6 12 12 --3 6 7 2 4 10 10

28 6 22 67 -54 61 125 91 18 22 159 31 8 7 -16 87 93 140 12 33 5 -11 9 13 19 51 --

26 6 22 58 -52 58 69 59 18 18 68 27 5 6 -9 60 60 45 12 21 5 -11 7 7 6 44 --

12 3 6 22 -19 21 25 21 10 10 25 12 5 6 -5 23 21 18 11 10 5 -6 7 5 5 18 --

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species are given in Table I. The numbers of loci, species, and genera from which these data were gathered are given in Table II. It can be seen from this table that although most of the enzymes considered here have been assayed from a wide range of species, some have been preferentially scored in either invertebrates or vertebrates. Perhaps the most obvious feature of Table I is that invertebrates generally show considerably higher levels of enzyme variability than vertebrates, confirming the earlier findings of Selander and Kaufman (1973). For the 21 enzymes scored in common in the two subkingdoms, invertebrates have a mean heterozygosity of 0.124 and vertebrates 0.046. This difference in means is highly significant (P

Relationship between enzyme heterozygosity and quaternary structure.

Biochemical Genetics, Vol. 15, Nos. 1/2, 1977 Relationship Between Enzyme Heterozygosity and Quaternary Structure Robert D. Ward 1 Received 18 May 1...
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