Papain-catalysed hydrolysis of some hippuric esters. A new mechanism for papain-catalysed hydrolyses

1. The Michaelis–Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k₀, is 2–3sec.⁻¹ and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k₀, less than 2–3sec.⁻¹. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme–substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis–Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, σ. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-α-amino acid derivatives.     

pH-dependence and structure–activity relationships in the papain-catalysed hydrolysis of anilides

The pH-dependence of the Michaelis-Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, K(s), is independent of pH between 3.7 and 9.3, whereas the acylation constant, k(+2), shows bell-shaped pH-dependence with apparent pK(a) values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett rho value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett rho values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.     

Dipeptidyl peptidase IV of human lymphocytes. Evidence for specific hydrolysis of glycylproline p-nitroanilide in T-lymphocytes.

Glycylproline p-nitroanilide is hydrolysed in lymphocytes from human blood exclusively by dipeptidyl peptidase IV. This was demonstrated by specific inhibition with N-alanylprolyl-O-(4-nitrobenzoyl)hydroxylamine and di-isopropyl phosphorofluoridate and by studying the membrane localization of dipeptidyl peptidase IV and determining specific dipeptidyl peptidase II activity. Additional evidence that dipeptidyl peptidase IV is a marker for T-lymphocytes, obtained from determinations of biochemical activity on intact lymphocyte preparations and correlation studies with other T-cell markers, is also presented.     

Cryoenzymology of trypsin. A detailed kinetic study of the trypsin-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at low temperatures.

A detailed study of the kinetics of the trypsin (EC 3.4.21.4)-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester in cryosolvents at 0 degrees C and below was undertaken. The pH-dependences of kcat, Km, k+2, k+3 and Ks were determined under cryoenzymological conditions and are compared with previous results [Antonini & Ascenzi (1981) J. Biol. Chem. 256, 12449-12455] obtained in fully aqueous media at ambient temperatures. Below pH 5.0 the kinetics, and presumably the mechanism of catalysis, are not significantly perturbed under cryoenzymological conditions. However, it is shown that below pH 5.0 both Km and Ks are decreased under these conditions but that both are increased at pH 6.7 relative to the results obtained in fully aqueous media at ambient temperatures. The effects of the cryoenzymological conditions on the individual catalytic parameters are discussed. The acylation rate constant, k+2, is essentially constant at pH 4.2 and 5.0 but decreases at lower pH values with an apparent pKa of approx. 4.0. In view of the low enthalpy of ionization associated with this pKa it is suggested that this group is the carboxy group of aspartic acid-189, which binds the positively charged lysine side chain of the substrate. The mechanistic implications of the results for the acylation step are discussed. It is also shown that only at low pH values can significant amounts of acylated trypsin be accumulated.     

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin.

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.     

Chemical synthesis of cellulose and cello-oligomers using a hydrolysis enzyme as a catalyst

Regio- and stereo-selective synthesis of polysaccharides and oligosaccharides has been achieved by using glycosyl fluorides as substrates for cellulases. This methodology has successfully been applied to the first synthesis of cellulose via a non-biosynthetic pathway as well as to a selective preparation of cello-oligosaccharides and unnatural oligosaccharides. Using the enzymatic polymerization, it is possible to control the relative direction (parallel or anti-parallel) of each glucan chain in the synthetic cellulose in vitro. Based on these results, a new concept of ‘allos-selectivity’ in polymer synthesis has been proposed.     

Modulation of lipase hydrolysis and synthesis reactions using carbohydrates.

A novel method for modulation of lipase hydrolysis and synthesis lipase was investigated by using carbohydrates in the microenvironment of the Candida rugosa enzyme. The influence of the addition of different sugars to the previously dialysed enzyme was tested on the two reactions. Rates of hydrolysis were lowered by using dialysed enzyme but were increased after sugar addition, regardless of the identity of the added sugar. In contrast, synthesis reaction rates depended on the nature of the carbohydrate. Rates were increased by adding lactose, which is not a water activity depressor, but were lowered by adding fructose, glucose, sucrose or sorbitol, which are all water activity depressors.     

The action of factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions

Kinetic parameters for the action of bovine Factor Xa (EC 3.4.21.22) on 25 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kc/Km, ranges from 1.5 X 10(1) to 2 X 10(6) M-1 X s-1 for the poorest and the best substates, respectively. The best substrates for Factor Xa were identified as those with arginine in the P1 position, and glycine in the P2 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effect of NaCl and CaCl2 concentrations and temperature on the action of Factor Xa on selected substrates have been assessed. Km values for Factor Xa hydrolysis of most substrates are greater than 100 microM. Solubility of the substrates consequently restricts measurements of reaction velocities to concentrations lower than desirable for optimally determining kc. Comparison of these kinetic parameters for Factor Xa with those of thrombin (Lottenberg, R., Hall, J.A., Blinder, M., Binder, E. and Jackson, C.M. (1983) Biochim. Biophys. Acta 742,539-557) for these same substrates indicates that the greater hydrolytic efficiency of thrombin is due primarily to lower Km values.     

Fermentative synthesis and hydrolysis of fructans

The review of the experimental papers dealing with some aspects of fermentative biosynthesis of fructans with different degrees of polymerization from microorganisms and plants is presented. The role of sucrose 1F-fructosyltransferase and 1,2-beta-fructan 1F-fructosyltransferase in the biosynthesis and hydrolysis of fructans, the influence of different stresses (water, temperature, oxygen and others) on these process are discussed.     

Chemical synthesis of hydroxymethylphosphonate alpha-DNA.

In this paper, we report the synthesis of hydroxymethylphosphonate alpha-DNAs and related compounds by use of the H-phosphonate method. These modified alpha-DNAs were designed to improve the inherent poor solubility of well-known methylphosphonate alpha-DNAs by introduction of a more hydrophilic hydroxymethylphosphonate function. The hybridization ability of hydroxymethylphosphonate alpha-DNAs was studied. We also report a novel strategy for the synthesis of alpha-thymidine by use of C1'-epimerization. The details of the neighboring effect of various 5'- and 3'-hydroxyl protective groups such as carbamoyl groups on the beta-->alpha conversion will be described.     

Chemical synthesis of all-trans-beta-retinoyl phosphat.

all-trans-beta-Retinoic acid is phosphorylated to retinoyl phosphate by bis(triethylamine) phosphate with yields of 10-15%. The product is soluble in methanol and is eluted from DEAE-cellulose acetate at a concentration of 0.1M-ammonium acetate in 99% (v/v) methanol. Its phosphate/retinoic acid molar ratio is 1. Retinoyl phosphate has an absorption maximum at 360nm in methanol, whereas retinoic acid has a maximum at 350 nm. The compound is hydrolysed at pH2 and pH13 for 20 min at 37 degrees C, but is relatively stable under the same conditions at pH4, 6, 8 and 10. Retinoyl phosphate (RF 0.1) can be separated from retinyl phosphate (RF 0.2) by chromatography on thin layers of silica gel in chloroform/methanol/water (60:25:4, by vol.).     

Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanilide substrates

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.     

Chemical synthesis of branched RNA.

A branched tetranucleotide consisting of adenosine linked 2' and 5' to guanosine and 3' to cytidine was synthesized from appropriately protected nucleoside phosphoramidites as synthons. The product was characterized enzymatically.     

Chemical synthesis and spontaneous glycosidic hydrolysis of 3-methyl-2''-deoxyguanosine and 2''-deoxywyosine [1]

3-methyl-2'-deoxyguanosine (1a) is obtained from 2'-deoxyguanosine by a reaction sequence involving conversion into tricyclic 1,N-2-isopropeno derivative (4-desmethyl-2'-deoxywyosine, 3a) followed by methylation which results in 2'-deoxywyosine (2a) and final removal of the 1,N-2 blocking system. Compounds 1a and 2a undergo spontaneous hydrolytic cleavage of their glycosidic bonds at pH 7, 37 degrees C, which makes them the most labile of all known nucleosides composed of structural units occurring in nature.     

Chemical methods of protein synthesis and modification.

Chemical and recombinant methods have continued to complement one another in the synthesis of protein analogues. Chemical methods remain particularly valuable when non-coded modifications are to be introduced, although it has been accepted since the commercialization of semisynthetic human insulin that they can also be used effectively for coded changes, in certain cases. The main objective of all such operations is not methodological, but is the production of molecules for practical use and further study. This goal has been reached frequently by chemical means during the past year.     

NAD hydrolysis: Chemical and enzymatic mechanisms

The pyridine nucleotide have important non-redox activities as cellular effectors and metabolic regulators [1–3]. The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD⁺ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to these many diverse biological activities. Included are the medically important NAD-dependent toxins associated with cholera, diphtheria, pertussis, and related disease [4]; the reversible ADPRibosylation-mediated biological regulatory systems [5,6]; the synthesis of poly (ADPRibose) in response to DNA damage or cellular, division [7]; and the synthesis of cyclic ADPRibose as part of an independent, calcium-mediated regulatory system[8]. As will be presented in this chapter, all evidence points to both the chemical and enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond being dissociative in character via an oxocarbenium intermediate.     

Thermal synthesis and hydrolysis of polyglyceric acid

Polyglyceric acid was synthesized by thermal condensation of glyceric acid at 80° in the presence and absence of two mole percent of sulfuric acid catalyst. The acid catalyst accelerated the polymerization over 100-fold and made possible the synthesis of insoluble polymers of both L- and DL-glyceric acid by heating for less than 1 day. Racemization of L-glyceric acid yielded less than 1% D-glyceric acid in condensations carried out at 80°C with catalyst for 1 day and without catalyst for 12 days. The condensation of L-glyceric acid yielded an insoluble polymer much more readily than condensation of DL-glyceric acid. Studies of the hydrolysis of poly-DL-glyceric acid revealed that it was considerably more stable under mild acidic conditions compared to neutral pH. The relationship of this study to the origin of life is discussed.