5 Lipolytic Enzymes
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H. BROCKERHOFF
1
Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia, Canada
A major group of lipolytic enzymes typified by pancreatic lipase consists of nonspecific esterases probably of the serine -histidine type. Another group, the phospholipases 2 of exo crine glands, are calcium activated and have exacting stereospecific substrate requirements. The enzymes of both groups hydrolyze water-insoluble esters. They must not only adsorb to oil—water or micelle—water interfaces but also must posi tion their active sites toward the matrix (oil droplet, micelle, or membrane) in which the substrate molecules are im bedded, the "supersubstrate." It is postulated that lipolytic enzymes are hydrolases that have developed supersubstrate binding sites for attachment and orientation toward lipids. Such a binding site, which is topographically distinct from the substrate binding sites of the reactive center, may have hydrophobic or electrostatic character.
A large part of the living matter on earth consists of lipids, and lipolytic enzymes play an important role in their biological turnover. Lipids can be classified into four major groups: (1) neutral esters of glycerol, especially triglycerides, (2) cholesterol and its relatives, (3) phospholipids, and (4) glycolipids. (The fourth group is not considered in this review). Table I shows the classification of some major lipolytic enzymes according to these categories. The enzymes are important be cause none of the fatty acid esters—triglycerides, cholesterol esters, or phospholipids—can be used for energy generation or in other metabolic reactions without prior enzymatic hydrolysis. Furthermore, the lipids cannot pass through the biological food chains except after hydrolysis; animals must hydrolyze fats to digest them. This fact makes lipolytic 1
Present address: Ν. Y. State Institute for Research in Mental Retardation, 1050 Forest Hill Rd., Staten Island, Ν. Y. 10314. 131 In Food Related Enzymes; Whitaker, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
132
FOOD R E L A T E D
Table I.
Groups of Lipids and Major Lipolytic Enzymes
Triglycerides: Lipases: Microbial Plants Milk Pancreatic Lipoprotein Hormone-sensitive Downloaded by OHIO STATE UNIV LIBRARIES on October 27, 2013 | http://pubs.acs.org Publication Date: October 1, 1974 | doi: 10.1021/ba-1974-0136.ch005
ENZYMES
Cholesterol Esters Cholesterol esterase Phospholipids Phospholipase 2 Phospholipase 1 Lysophospholipase
enzymes interesting for the nutritionist. In addition, many practical problems in food processing involve lipolytic enzymes. The enzymes that hydrolyze triglycerides are lipases (Table I ) . Many microorganisms such as molds and bacteria produce these enzymes. Lipases may cause spoilage of food but may also be beneficial. The aroma and flavor of Roquefort cheese, for example, is partly a result of the hydrolysis of milk fat by the lipase of a mold. The characteristic flavor of many Italian cheeses, on the other hand, is produced by pregastric lipase which is added to the milk as an extract from the calf stomach. Plant lipases are found in the seeds. They are needed during germination when the seed oils are degraded to provide energy for rapid growth. They cause hydrolysis of an o i l during extraction, and this re quires additional production steps. M i l k contains at least two lipases. One of them is activated by foaming which can be caused by air leaks in the pipelines that carry fresh, raw milk. The result is a rancid flavor caused by free short-chain fatty acids such as butyric acid. Three lipases are important to man and higher animals: (a) pancreatic lipase, which digests triglycerides to fatty acids and monoglycerides, which can then pass through the intestinal wall, (b) lipoprotein lipases, which degrade the triglycerides that circu late i n the blood; there are at least two different enzymes of this kind, one produced in the liver and one i n other tissues ( 1 ), (c) hormone-sensitive lipase, which is triggered by adrenalin to hydrolyze and mobilize the depot fat of adipose tissue. Lipases can be divided into two groups according to their positional specificity. Some lipases hydrolyze only the ester bonds in positions 1 and 3 of glycerol, i.e., the primary esters. This is true for pancreatic lipase (Table I I ) . Other lipases, such as many microbial lipases, hydro lyze a l l three ester bonds. In this case, the primary esters are probably hydrolyzed faster than the secondary ester. The best-known cholesterol esterase (Table I) is also a pancreatic enzyme. Cholesterol esterases not only hydrolyze cholesterol esters but also synthesize them from cholesterol and fatty acid in a reversible reac tion. This is important i n the digestion of cholesterol itself because
In Food Related Enzymes; Whitaker, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
5.
BROCKERHOFF
Lipolytic Enzymes
133
cholesterol is esterified in the intestinal wall before it is incorporated into chylomicrons and delivered into the blood. Cholesterol esterase is a very unspecific enzyme which w i l l also hydrolyze monoglycerides and watersoluble esters (2). Many of the phospholipases, on the other hand, are very selective enzymes. The pancreatic phospholipase 2 (or phospholipase A ) hydrolyzes only esters in position 2 of sn-3 phospholipids; i.e., the enzyme is stereospecific (Table II). Phospholipases I (or A ) attack only the 1-position. Lysophospholipases remove the remaining fatty acid, either in position 1 or 2, from the lysophospholipid. Phospholipases play a role in the post-mortem degradation of meat and fish. In the remainder of the paper, I concentrate on the two best-known lipolytic enzymes—pancreatic lipase and phospholipase 2—and then speculate on the special nature of lipolytic reactions and how lipolytic enzymes differ from hydrolases. 2
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x
Table II.
Positional Specificity of Pancreatic Enzymes Ο
0
2 R—CO—I
Lipase
sw-position 1
L
+2FA
-OH
OC—R
Ο II
Ο
Ο
rOH
R—CO—I
Ο
3
2
Ο
rOC—R
r-OC
R
II
II
R—CO- OC—R Ο n
L
OPO—X
Phospholipase
HO—
ο
II
-ΟΡΟ—X
oPancreatic
Lipase
The most thoroughly investigated pancreatic lipase is from the pig. Most of our knowledge of the structure and properties of this enzyme comes from P. Desnuelle and his co-workers in Marseille (3) (Table III). The lipase occurs in two similar forms, isoenzymes Lipase A and Lipase B. There is no known proenzyme (zymogen). The enzyme does not react with diisopropylfluorophosphate ( D F P ), the standard inhibitor
In Food Related Enzymes; Whitaker, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
134
FOOD R E L A T E D
Table III.
ENZYMES
Molecular Properties of Porcine Pancreatic Lipase
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Molecular weight 48,000 N o proenzyme Amino acid sequence: leu-ser-gly-his Reactive residues: ser, his Carbohydrates: mannose4, iV-acetylglucosamine3 of esterases that have serine as a reactive amino acid. It does react with a similar inhibitor, diethyl-p-nitrophenyl phosphate ( D N P ) (4). Beside serine, a histidine is a reactive residue. The lipase is probably a serinehistidine enzyme like many other esterases and proteinases such as chymotrypsin or elastase. The amino acid sequence around the reactive serine is remarkable. Where other enzymes have glycine or an acidic residue such as aspartic acid, lipase has leucine. The total amino acid sequence of the lipases is not yet known. There are four or five disulfide bridges in the molecule and two free S H groups which are not involved in the catalytic mechanism (5). The carbohydrate residues are less than 2 % of the molecule ( 6 ) and have no obvious function. The substrate specificity of lipase has been investigated in my labora tory. A n ester such as a glyceride is likely to be hydrolyzed by nucleophilic attack:
Ο
II R—O—C—R Î X
1
The chemistry of such reactions is well understood. The attack is facilitated by withdrawal of electrons from the carbonyl carbon. This can be achieved by electrophilic substituents in R. The electron-with drawing power is expressed as the Hammett constant (Table I V ) . If this constant is positive, the substituent is electron withdrawing rela tive to hydrogen (7). The inductive power increases with increasing electronegativity of the substituent and is quite strong in ester groups. When esters of different alcohols are hydrolyzed by pancreatic lipase (Table V ) and the maximal reaction rates are compared the inductive effect of the substituent is also apparent (8). The reaction proceeds by nucleophilic attack. Triglycerides are good substrates for the lipase be cause each of their ester groups is activated by two other ester groups. A second factor in nucleophilic reactions is steric hindrance. Table V I shows how the change from primary to secondary to tertiary ester influences the rate of alkaline hydrolysis of an ester (7). A parallel
In Food Related Enzymes; Whitaker, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
5.
BROCKERHOFF Table IV. CH
OH
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Hammett Constants σ of Electrophillic Induction -0.07
3
-0.002
OCH.
135
Lipolytic Enzymes
+0.12
„ (^J
+0.22
F
+0.34
CI
+0.37
0 II R-C-0
CN
(7)
+0.40
+0.68
Ν-
+0.71
0*
8
relationship between steric hindrance and reaction rates is observed when different esters of oleic acid are hydrolyzed by lipase ( Table V I I ) (8). Beta-monoglycerides (2-acyl-glycerols) are resistant to pancreatic lipase. This resistance and the corresponding specificity of the lipase for the primary positions can be explained by steric hindrance. A third factor regulating the speed of lipolysis is the hydrophobicity of the ester. The normal substrates of lipase—the natural triglycerides— are insoluble in water, and the enzyme acts at the oil-water interface. Thus, lipases have been defined as esterases that act on insoluble sub strates at such interfaces. However, triglycerides that are similar i n steric and inductive effects may still react with different velocities even if all reactions take place at oil-water interfaces. For instance, emulsified tributyrin is hydrolyzed 20 times faster than emulsified triacetin. This difference is caused by the different hydrophobicity of the triglycerides not by the different chain lengths of butyric and acetic acid. When comparing the reaction rates of a series of esters of similar structure (9) (Figure 1), it is seen that the esters of highest solubility— Table V. Relative Maximal Rates V of Hydrolysis of Oleic Acid Esters by Pancreatic Lipase, Compared with Triolein, V = 1.0 (7) H0-—· — 0 -
0.05
0 II RC0-»—-0-
WW-0-
0.08
ρ——0-
0.30
0.07
φ-.-Ο-
0.27
NC-«-0-
0.80
CH -03
VW>——-0-
0.16
027
c l
Br-——0-
0.16
ci-—»-0-
0.25
O~
N0 Q-—02
1
0
>I0
In Food Related Enzymes; Whitaker, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1974.
136
FOOD R E L A T E D
Table VI.
Relative Rates of Alkaline Hydrolysis of Methyl, Ethyl, Isopropyl, and tert-Butyl Acetate (7) 0 II
•-0-C —· 0 H • - • - Ο - C - · Downloaded by OHIO STATE UNIV LIBRARIES on October 27, 2013 | http://pubs.acs.org Publication Date: October 1, 1974 | doi: 10.1021/ba-1974-0136.ch005
ENZYMES
.
1.00
0 II
\
\_o_c_. .
0.60
0 ii - ρ Ο - C - ·
o.l5
Τ
0.008
Table VII. Relative Rates V of Hydrolysis of Sterically Hindered Oleic Acid Esters by Pancreatic Lipase, Compared with Triolein, V = 1.0 (8)
F
\ - 0 -
